WO2020156048A1 - 利用基于超声原理的动植物组织消融仪快速提取rna的方法 - Google Patents

利用基于超声原理的动植物组织消融仪快速提取rna的方法 Download PDF

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WO2020156048A1
WO2020156048A1 PCT/CN2020/070559 CN2020070559W WO2020156048A1 WO 2020156048 A1 WO2020156048 A1 WO 2020156048A1 CN 2020070559 W CN2020070559 W CN 2020070559W WO 2020156048 A1 WO2020156048 A1 WO 2020156048A1
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unit
voltage
animal
ablation instrument
plant tissue
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PCT/CN2020/070559
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English (en)
French (fr)
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赵仲玖
廖东升
邱坤
赵伟
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成都导胜生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the invention belongs to the technical field of biological preparations, and specifically relates to a method for rapidly extracting RNA by using an animal and plant tissue ablation instrument based on the principle of ultrasound.
  • RNA is often isolated and purified from tissues and cells, and the quality of RNA often affects the success or failure of molecular biology experiments such as cDNA library, RT-PCR, and Northern blot.
  • the commonly used method is to first protect the tissue with tissue protection solution, and then use the extract solution to extract RNA from the tissue or cell.
  • Commonly used protection methods include liquid nitrogen protection and protection solution protection, and the commonly used protection solutions include RNA later reagent and RNA safety reagent.
  • the liquid nitrogen protection method not only requires harsh conditions, but also has a high cost, which is only suitable for temporary tissue protection.
  • the protective solution usually needs to be stored at low temperature, and the RNAlater solution is not compatible with the chemical components of TRIzol, silica gel column and magnetic bead method.
  • the RNAlater solution contains ammonium isothiocyanate.
  • tissue block is from the RNAlater solution
  • the ammonium isothiocyanate in the RNAlater solution will bind or react with the phenol and SDS in the extract to form a precipitate, resulting in The composition and concentration of the extract change, and the lysis is not complete, which reduces the purity and yield of RNA. Therefore, tissues or cells preserved with RNA later solution usually need to be washed repeatedly before extraction, which wastes time and manpower and reduces extraction efficiency.
  • RNA extraction often uses TRIzol method, silica gel column, magnetic bead method, etc., but these methods generally suffer from low RNA yield and serious contamination of impurities, which cannot meet the quality of subsequent experiments such as genetic diagnosis, biochip analysis, and gene expression analysis. Claim.
  • RNA extraction from animal-derived tissue blocks has difficulties such as complicated operations, incomplete fragmentation, and difficulty in inhibiting endogenous RNA activity, which makes it difficult to guarantee the quality and yield of RNA extraction.
  • the purpose of the present invention is to provide a method for rapidly extracting RNA using an animal and plant tissue ablation instrument based on the principle of ultrasound.
  • the method for rapidly extracting RNA by using an ultrasound-based ablation instrument for animal and plant tissues includes steps:
  • the extract A includes 1.2-2.0mol/L guanidine thiocyanate and 0.5-1.5mol/L guanidine hydrochloride, and the pH is 4;
  • extract B added to the tissues or cells preserved in extract A, and mix upside down to obtain a mixture.
  • the extract B includes 30-65vt% water-saturated phenol and 9-15vt% in volume fraction Glycerin, the balance is solvent;
  • Extraction solution B also includes 0.20-1mol/L guanidine thiocyanate, 0.4-0.8mol/L ammonium thiocyanate and 0.0073-0.018mol/L surfactant in molar concentration, and the pH is adjusted to 4;
  • step G3 When extracting RNA from tissues, the mixture obtained in step G2 is ultrasonically disrupted with an animal and plant tissue ablation instrument based on the principle of ultrasound; when RNA is extracted from cells, directly perform the following operations in step G5;
  • the animal and plant tissue ablation instrument based on the ultrasound principle includes: a power supply, a DCDC conversion unit, a main control unit, a DCDC power adjustment unit, a drive unit, a transformer unit, a resonance unit, an ultrasonic transducer, and a sampling unit;
  • the power supply is used by the animal and plant tissue ablation instrument after the voltage is converted by the DCDC conversion unit and the DCDC power adjustment unit; the main control unit outputs the first PWM signal to control the DCDC power adjustment unit to output an adjustable voltage, and collects the voltage of the transformer unit Feedback from the sampling unit of the output loop voltage and current, output two second PWM signals with complementary duty cycles to the drive unit and undergo transformation by the transformer unit, and then pass through the resonance unit set in the output loop of the transformer unit The resonance point is adjusted to the resonance point of the ultrasonic vibrator, so that the ultrasonic vibrator arranged in the output loop of the transformer unit works in a resonance state.
  • the ultrasonic vibrator includes an ultrasonic transducer and an horn that transmits the vibration input from the ultrasonic transducer after changing the amplitude.
  • the surfactant in the extract B is sodium lauryl sulfate.
  • the extract solution A uses hydrochloric acid to adjust the pH value
  • the extract solution B uses acetic acid to adjust the pH value.
  • the extract B also includes ⁇ -mercaptoethanol with a molar concentration of 6.4 mmol/L to 19.20 mmol/L.
  • the extract B also includes potassium citrate with a molar concentration of 0.05-0.15 mmol/L.
  • the extract B also includes bromophenol blue with a molar concentration of 74.62 ⁇ mol/L-223.87 ⁇ mol/L.
  • the extract B also includes sodium acetate with a molar concentration of 0.01-2 mol/L.
  • the extraction liquid B also includes isopropanol with a volume fraction of 5-25%.
  • the extract B also includes 8-hydroxyquinoline with a molar concentration of 0.689 mmol/L to 68.9 mmol/L.
  • the extract B also includes DTT with a molar concentration of 0.5-1000 mmol/L.
  • the extract B also includes urea with a molar concentration of 0.5-5 mol/L.
  • the extract B also includes EDTA with a molar concentration of 0.1-500 mmol/L.
  • the extract B also includes ammonium acetate with a molar concentration of 0.01-10 mmol/L.
  • the water in the step G8 is double distilled water, deionized water or ultrapure water.
  • the total mass of the extract A and the extract B is 100-150 times the mass of the tissue or cell.
  • the ultrasonic power is 0.5-1000W
  • the ultrasonic time is 0.5-30s.
  • the volume of chloroform is 0.1-0.4 times the volume of the mixture.
  • the volume ratio of isopropanol to the upper liquid is 1:1.
  • the main components of the extract of the present invention are guanidine thiocyanate and guanidine hydrochloride.
  • the guanidine thiocyanate is an organic compound with a molecular formula of C 2 H 6 N 4 S, which is mainly used in biomedicine and chemical reagents.
  • guanidine thiocyanate is a decoupling agent, a powerful protein denaturant, and destroys the secondary structure of proteins; it mainly plays a role in cell lysis and promotes the separation of nucleoprotein and nucleic acid; and acts as an enzyme inhibitor with a tissue preservation effect.
  • the guanidine hydrochloride is a white or slightly yellow mass, which is used as a strong denaturant for extracting total cellular RNA.
  • Guanidine hydrochloride solution can dissolve protein, cause cell structure damage, nuclear protein secondary structure damage, and dissociate from nucleic acid.
  • RNase can be inactivated by reducing agents such as guanidine hydrochloride.
  • As a nuclease inhibitor it can be Extract RNA from tissues containing RNase.
  • guanidine thiocyanate can lyse tissues or cells, but at the same time, the increased guanidine hydrochloride can inhibit the activity of RNase to achieve the effect of lysis and preservation.
  • guanidine thiocyanate and guanidine hydrochloride Through the quantitative configuration of guanidine thiocyanate and guanidine hydrochloride, it can provide tissues or cells with a certain period of room temperature preservation effect, and generally can be stored at room temperature for more than 7 days.
  • Sodium dihydrogen phosphate, sodium chloride and magnesium chloride can be used in the extraction solution as a buffer system to maintain the concentration of salt ions.
  • the water-saturated phenol contained therein has the effect of lysing cells and precipitating proteins, and helps to remove DNA when it is acidic.
  • Ammonium thiocyanate is a colorless crystal, easily deliquescent, soluble in water and ethanol, and soluble in methanol and acetone.
  • the surfactant uses SDS, namely sodium dodecyl sulfate, which has the effect of denaturing proteins; and by adjusting the pH value To 3.5-4.5 is mainly to ensure the quality of extraction, because too low pH will mainly reduce the purity of extraction, while too high will affect the yield.
  • ⁇ -mercaptoethanol which has the functional groups of ethylene glycol and ethanedithiol, is a volatile liquid with a strong pungent odor.
  • ⁇ ME is usually used for the reduction of disulfide bonds and can be used as an antioxidant in biological experiments.
  • the reason why it is widely used is that its hydroxyl group enables it to be dissolved in water and reduces its volatility. In the present invention, it mainly serves to destroy the disulfide bond in RNase, thereby having an inhibitory effect.
  • Increased potassium citrate is a commonly used buffer. It is a white, slightly hygroscopic crystalline powder. Odorless, with the taste of saline, soluble in water, slowly soluble in glycerin, insoluble in alcohol, salty and cool in taste. It acts as a stabilizer and pH buffer, etc., participates in the buffer system, and acts as an enzyme inhibitor.
  • the molecular formula of the added bromophenol blue is C 19 H 10 Br 4 O 5 S, which is a light yellow to brownish yellow powder, and is usually used as an electrophoresis indicator dye, and is also used as a color indicator in the formulation of the present invention.
  • bromophenol blue sinks with DNA and protein, which can be used to indicate whether RNA isolation is complete.
  • the increased sodium acetate can precipitate sugars in the solution during the RNA extraction process, thereby achieving the effect of removing impurities and increasing the purity of the extracted RNA.
  • the added isopropanol is an organic compound, a colorless and transparent liquid, which protects the hydrophilic groups in the RNA chain through the hydrophobic effect of -OH, and has the effect of reducing the upper layer RNA.
  • Increased 8-hydroxyquinoline can also inhibit RNase, and the combined use with chloroform can enhance the inhibitory effect.
  • the increased DTT is a reducing agent, which is consistent with the effect of mercaptoethanol, which mainly destroys the disulfide bond in RNase, thereby having an inhibitory effect.
  • Increased urea is a white crystal-like organic compound; high-concentration urea can denature protein and can also inhibit RNase activity.
  • Increased EDTA is a divalent metal chelate and also acts as an enzyme inhibitor.
  • the structural formula of the added ammonium acetate is CH 3 COONH 4 , also known as ammonium acetate. It is a kind of white triangular crystal with acetic acid smell, which can be used as analytical reagent and meat preservative.
  • the protein encountered in RNA extraction is precipitated, but the ammonium ion easily forms a white precipitate with the thiosulfate ion.
  • the extract A can store the tissue at room temperature for 24 hours to ensure that the RNA is not degraded, that is, it does not need to be stored at a low temperature and is free from the cold chain.
  • the invention adopts ultrasonic to break the tissue, and the speed is fast, which not only avoids incomplete tissue fragmentation and the interference of endogenous RNase, but also greatly shortens the extraction time and further improves the extraction efficiency of RNA.
  • the DCDC power adjustment unit of the ultrasound-based animal and plant tissue ablation instrument includes: a switch control signal receiving end connected to a main control unit to receive switch control signals Signal receiving circuit;
  • the first PWM signal receiving end is connected to the first PWM signal receiving circuit that the main control unit receives the first PWM signal
  • the power is converted into a fourth DCDC conversion unit for adjustable voltage output through the switch control signal and the first PWM signal sent by the main control unit.
  • the fourth DCDC conversion unit of the animal and plant tissue ablation instrument includes: a fourth voltage input filter that sequentially processes the power supply voltage, a fourth voltage conversion chip U1, and a fourth voltage output filter, and the fourth voltage input filter includes The filter capacitor at the power supply terminal, the upper tube drive signal reference point pin SW of the fourth voltage conversion chip U1 is connected to the adjustable voltage output terminal through the energy storage inductor L1, and the voltage divider resistors R32 and R47 are connected in series to the adjustable voltage output terminal for voltage sampling Input to the reference voltage pin FB of the fourth voltage conversion chip U1, the upper tube drive signal reference point pin SW of the fourth voltage conversion chip U1 is also connected with a freewheeling diode D2, and the enable pin EN is also connected with a resistor R33, Resistor timing/external clock pin RT/CLK is also connected with a frequency divider resistor R85, and frequency compensation pin COMP is also connected with capacitors C51, C49 and resistor R46 for regulating the loop and stabilizing voltage output.
  • the switch control signal receiving circuit includes: resistors R104 and R105 connected in series between the converted voltage and the switch control signal receiving end, the emitter is connected to the converted voltage, the base is connected to the node between the resistors R104 and R105, and the collector is connected to the fourth The transistor Q12 of the enable pin EN of the voltage conversion chip U1, when the switch control signal is L, the transistor Q12 is turned on, and the fourth voltage conversion chip U1 is turned on;
  • the first PWM signal receiving circuit includes: RC filtering and a first voltage follower U3B that sequentially process the first PWM signal, the first PWM signal received by the first PWM signal receiving end is filtered by the RC filter and then input to the first voltage follower
  • the non-inverting input terminal of U3B and the output terminal of the first voltage follower U3B are connected to the node between the voltage dividing resistors R32 and R47 through the voltage dividing resistor R54.
  • the animal and plant tissue ablation instrument further includes an output voltage sampling unit for collecting the adjustable voltage of the DCDC power adjustment unit, and the collected adjustable voltage is sent to the main control unit through the second voltage follower U3A.
  • the driving unit of the animal and plant tissue ablation instrument includes a first driving unit and a second driving unit, and the second PWM signal receiving terminal N of the first driving unit receives the first second PWM signal sent by the main control unit,
  • the output terminal of the first driving unit is connected to the end of the same name of the primary coil of the transformer unit, and the second PWM signal receiving terminal P of the second driving unit receives the second PWM signal sent by the main control unit.
  • the output terminal is connected to the synonymous terminal of the primary coil of the transformer unit, and the first driving MOS transistor Q6 is controlled to turn on/off through the first second PWM signal received by the second PWM signal receiving terminal N, and is received through the second PWM signal
  • the second second PWM signal received by the terminal P controls the second driving MOS transistor Q2 to turn off/on.
  • the transformation unit of the animal and plant tissue ablation instrument is a push-pull transformer
  • the adjustable voltage output end of the DCDC power adjustment unit is connected to the synonymous end of the first coil and the same name end of the second coil of the primary side of the push-pull transformer ;
  • the first driving unit includes resistors R10 and R14 connected in series between the ground and the second PWM signal receiving terminal N, the gate is connected to the node between the resistors R10 and R14, the source is grounded, and the drain is the output of the first driving unit Terminal and connected to the first driving MOS transistor Q6 of the same name terminal of the first coil of the primary side of the push-pull transformer;
  • the second driving unit includes resistors R5 and R13 connected in series between the ground and the second PWM signal receiving terminal P, the gate is connected to the node between the resistors R5 and R13, the source is grounded, and the drain is the output of the second driving unit
  • the second driving MOS transistor Q2 of the alias end of the second coil of the primary side of the push-pull transformer is connected in parallel.
  • the first driving MOS transistor Q6 When the first second PWM signal received by the second PWM signal receiving terminal N is H and the second PWM signal received by the second PWM signal receiving terminal P is L, the first driving MOS transistor Q6 is controlled to be turned on, The second driving MOS tube Q2 is turned off, the input loop of the first coil of the primary side is connected and the input current direction is from the synonymous end of the first coil of the primary side to the same name end, the input loop of the second coil of the primary side is turned off, and the secondary side The output current direction of the output loop of the coil is from the same name end to the different name end of the secondary coil.
  • the first driving MOS transistor Q6 is controlled to be turned off, and the first The second drive MOS tube Q2 is turned on, the input loop of the second coil of the primary side is connected and the input current direction is from the same name end of the second coil of the primary side to the different name end, the input loop of the first coil of the primary side is cut off, and the secondary side
  • the output current direction of the output loop of the coil is from the different-named end of the secondary coil to the same-named end.
  • the input DC voltage is converted into an AC waveform with a frequency of 30KHz and a voltage of hundreds of volts, which provides conditions for the subsequent resonant unit.
  • the resonance unit of the animal and plant tissue ablation instrument is LC series resonance
  • the LC series resonance includes an inductance T1 and a capacitor C1 connected in series in the output loop of the transformer unit.
  • the ultrasonic transducer of the animal and plant tissue ablation instrument is arranged in the output loop of the transformer unit through the interface J2.
  • the sampling unit of the animal and plant tissue ablation instrument includes a voltage sampling unit that collects the output loop voltage of the transformer unit and a current sampling unit that collects the output loop current of the transformer unit.
  • the output of the transformer unit The end loop is provided with multiple sampling resistors connected in series; the voltage signal is collected through the voltage sampling terminal connected to the node between the multiple sampling resistors, then sent to the voltage sampling unit for filtering and amplification, and then sent to the main control unit for resonance adjustment;
  • the current sampling end of the output loop connected in series with the transformer unit collects the current signal and sends it to the current sampling unit for filtering and amplification, and then to the main control unit for resonance adjustment.
  • the main control unit of the animal and plant tissue ablation instrument includes a main control chip U2 and a main control chip peripheral circuit.
  • the main control chip U2 outputs a first PWM signal to control the DCDC power adjustment unit to output an adjustable voltage, and collect and transform the voltage
  • the feedback of the sampling unit of the output loop voltage and current of the unit outputs two second PWM signals with complementary duty cycles to the driving unit.
  • the main control unit further includes an auxiliary chip U1 and auxiliary chip peripheral circuits.
  • the main control chip U2 of the main control unit sends feedback to the auxiliary chip U1 by collecting feedback from the sampling unit of the output loop voltage and current of the transformer unit Instruction, the auxiliary chip U1 receives the instruction from the main control chip U2 and outputs two second PWM signals with complementary duty cycles to the driving unit. At the same time, the two second PWM signals with complementary duty cycles are also fed back to the main control chip U2.
  • the two second PWM signals with complementary duty cycles are further amplified by the first signal amplifier output circuit and the second signal amplifier output circuit, respectively, and then sent to the driving unit.
  • the animal and plant tissue ablation instrument further includes a display unit connected to the main control chip U2, and the display unit includes a display chip J8 and an LCD display screen connected to the display chip J8.
  • the animal and plant tissue ablation instrument further includes a button connected to the main control chip U2.
  • the animal and plant tissue ablation instrument further includes a memory connected to the main control chip U2.
  • the animal and plant tissue ablation instrument further includes a USB interface unit connected with the main control chip U2, and the USB interface unit includes a USB chip ESD1 connected with the main control chip U2 and an interface J7 connected with the USB chip ESD1.
  • the animal and plant tissue ablation instrument further includes a touch unit connected to the main control chip U2.
  • the extract A of the present invention can replace the traditional RNA later solution for preserving tissues or cells. Therefore, when the extract A and the extract B are used to extract RNA, there is no need to wash the tissues or cells, which saves you
  • the cleaning operation when storing RNA later solution simplifies the operation steps and significantly improves the extraction efficiency;
  • the animal and plant tissue ablation instrument of the present invention uses the first PWM signal to control the DCDC power adjustment unit to output an adjustable voltage, so that the power is adjustable from 0.5 to 1000W, and uses the sampling of the output loop voltage and current of the acquisition transformer unit
  • the feedback of the unit, output two second PWM signals with complementary duty cycles to the drive unit make the resonant ultrasonic frequency adjustable from 20-200KHz, adjust the resonance point to the resonance point of the ultrasonic vibrator, make the ultrasonic vibrator work in the resonance state, and output
  • the energy is the largest, the amplitude is the strongest, the ultrasonic is used to break the tissue, and the speed is fast, which not only avoids incomplete tissue fragmentation and endogenous RNase interference, but also greatly shortens the extraction time and further improves the RNA extraction efficiency;
  • the properties of the extract A and the extract B of the present invention are relatively stable, are not easily affected by conditions such as temperature and humidity, and can be fully used in conjunction to achieve the purpose of rapid, low-cost, high-quality, and large-scale extraction of animal RNA in the laboratory;
  • the newly added guanidine hydrochloride of the present invention has the effect of a nuclease inhibitor and can effectively extract RNA from RNase-rich tissues.
  • the new sodium acetate has the effect of precipitating sugars encountered in RNA extraction, ensuring the purity of the extracted RNA.
  • Newly added isopropanol through the hydrophobic effect of -OH, the hydrophilic group in the RNA chain is protected, and at the same time has the effect of reducing the upper layer RNA;
  • the present invention adjusts the amount of reagents for preservation of tissues in solution A, and the tissues can be stored at room temperature for 10 days or more, but for the liver tissues rich in RNase, the results of RNA extracted are normal, and the yield is improved; In addition, it mainly increases multiple components in RNase pollution to prevent RNA from being degraded.
  • Fig. 1 is a system block diagram of an embodiment of the present invention.
  • Fig. 2 is a circuit principle diagram of the power supply, DCDC conversion unit and DCDC power adjustment unit of the control board of the embodiment of the present invention.
  • Fig. 3 is a circuit diagram of the main control unit of the control board of the embodiment of the present invention.
  • Fig. 4 is a circuit principle diagram of the sampling unit, the display unit and the auxiliary chip of the control board of the embodiment of the present invention.
  • Fig. 5 is a circuit diagram of the interface of the control board of the embodiment of the present invention.
  • Fig. 6 is a circuit principle diagram of the driving unit, the transformer unit, and the resonance unit of the driving board of the present invention-embodiment.
  • Figure 7 is a picture of agarose gel electrophoresis in Example 5 of the present invention.
  • the animal and plant tissue ablation instrument based on the ultrasound principle of this embodiment includes a power supply, a DCDC conversion unit, a main control unit, a DCDC power adjustment unit, a drive unit, a transformer unit, a resonance unit, and an ultrasonic converter. Energy detector and sampling unit.
  • the power supply is used by the DCDC conversion unit and the DCDC power adjustment unit to convert the voltage to the animal and plant tissue ablation instrument; the main control unit outputs the first PWM signal to control the DCDC power adjustment unit to output an adjustable voltage, and collects the output terminal of the transformer unit Feedback from the sampling unit of loop voltage and current, output two second PWM signals with complementary duty cycles to the drive unit and undergo voltage transformation by the transformer unit, and then adjust the resonance through the resonance unit provided in the output loop of the transformer unit Point to the resonance point of the ultrasonic vibrator, so that the ultrasonic vibrator arranged in the output loop of the transformer unit works in a resonance state.
  • the animal and plant tissue ablation instrument is further elaborated.
  • the power supply is a DC24V power supply
  • the DCDC conversion unit includes a first DCDC conversion unit, a second DCDC conversion unit, and a third DCDC conversion unit.
  • the first DCDC conversion unit includes TVS protection, first voltage input filtering, first voltage conversion chip, and first voltage output filtering.
  • the 24V DC power supply sequentially passes through TVS protection, first voltage input filtering, first voltage conversion chip and first The voltage output is filtered and converted into 12V output voltage, which is supplied to the main control unit and drive unit.
  • the DC24V power supply passes through the protective diode TVS1 in turn for TVS protection, through three capacitors C59, C60 and C57 connected in parallel to the DC24V power supply terminal for voltage input filtering, and then passes through the voltage divider connected in series to the DC24V power supply terminal
  • the resistors R48 and R51 perform voltage sampling and input the enable pin EN of the first voltage conversion chip U6, the model of the first voltage conversion chip U6 is TPS54340, and the upper tube drive signal reference point pin SW of the first voltage conversion chip U6 is stored
  • the inductance L2 is connected to the 12V output voltage terminal, and the voltage divider resistors R88 and R89 connected in series to the 12V output voltage terminal are used for voltage sampling and input to the reference voltage pin FB of the first voltage conversion chip U6, through the capacitor C72 connected to the 12V output voltage terminal.
  • the upper tube drive signal reference point pin SW of the first voltage conversion chip U6 is also connected with a freewheeling diode D4, the enable pin EN is also connected with a soft start capacitor C79, resistor timing/external clock pin RT /CLK is also connected with a frequency divider resistor R50, and the frequency compensation pin COMP is also connected with capacitors C61, C62 and a resistor R52 for regulating the loop and stabilizing voltage output.
  • the capacitor C62 is connected in parallel with the capacitor C61 and the resistor R52 in series.
  • the second DCDC conversion unit includes a second voltage input filter, a second voltage conversion chip, and a second voltage output filter.
  • the 12V input voltage is sequentially converted to 1.8V through the second voltage input filter, the second voltage conversion chip and the second voltage output filter.
  • the output voltage is supplied to the main control unit.
  • the 12V input voltage passes through two capacitors C84 and C85 connected in parallel to the 12V input voltage terminal for voltage input filtering.
  • the model of the second voltage conversion chip U9 is TLV62130ARGTR, and the second voltage conversion chip U9
  • the MOS tube drive signal reference point pin SW is connected to the 1.8V output voltage terminal through the energy storage inductor L3, and the voltage divider resistors R90 and R91 connected in series to the 1.8V output voltage terminal are used for voltage sampling and input to the reference voltage of the second voltage conversion chip U9
  • the pin FB is filtered by capacitors C80 and C83 connected in parallel to the 1.8V output voltage terminal.
  • the internal power supply pin PVIN of the second voltage conversion chip U9, the internal control circuit power supply pin AVIN, and the enable pin EN are also connected At the 12V input voltage terminal, the soft start/tracking pin SS/TR is also connected with a soft start capacitor C86, the adjustment output pin DEF and the frequency configuration pin FSW are both grounded, and the output voltage acquisition pin VOS is connected to the 1.8V output voltage terminal.
  • the third DCDC conversion unit includes a third voltage input filter, a third voltage conversion chip, and a third voltage output filter.
  • the 12V input voltage is sequentially converted to 3.3V through the third voltage input filter, the third voltage conversion chip and the third voltage output filter.
  • the output voltage is supplied to the main control unit and sampling unit.
  • the circuit principle of the third DCDC conversion unit is elaborated in detail: the 12V input voltage passes through two capacitors C89 and C90 connected in parallel to the 12V input voltage terminal for voltage input filtering.
  • the third voltage conversion chip U10 is modeled as TLV62130ARGTR, and the third voltage conversion chip U10
  • the MOS tube drive signal reference point pin SW is connected to the 3.3V output voltage terminal through the energy storage inductor L4, and the voltage divider resistors R92 and R94 connected in series to the 3.3V output voltage terminal are used for voltage sampling and input to the reference voltage of the third voltage conversion chip U10
  • the pin FB filters the voltage output through capacitors C87 and C88 connected in parallel to the 3.3V output voltage terminal.
  • the internal power supply pin PVIN of the third voltage conversion chip U10 and the internal control circuit power supply pin AVIN are also connected to the 12V input voltage terminal.
  • the voltage divider resistors R93 and R102 connected in series with the 12V input voltage terminal perform voltage sampling and input to the enable pin EN of the third voltage conversion chip U10.
  • the enable pin EN of the third voltage conversion chip U10 is also connected to the second voltage conversion chip U9 When the second DCDC conversion unit does not work normally, the third DCDC conversion unit stops working.
  • the soft-start/tracking pin SS/TR is also connected with a soft-start capacitor C91 to adjust the output pin. DEF and the frequency configuration pin FSW are both grounded, and the output voltage acquisition pin VOS is connected to the 3.3V output voltage terminal.
  • the DCDC power adjustment unit includes a switch control signal receiving circuit, a first PWM signal receiving circuit, and a fourth DCDC conversion unit.
  • the fourth DCDC conversion unit includes a fourth voltage input filter, a fourth voltage conversion chip, and a fourth voltage output filter.
  • the DCDC conversion unit converts the DC24V power supply into a 0-24V adjustable voltage output through the switch control signal and the first PWM signal sent by the main control unit.
  • the circuit principle of the fourth DCDC conversion unit The DC24V power supply sequentially passes through three capacitors C6, C7 and C41 connected in parallel to the DC24V power supply terminal for voltage input filtering.
  • the fourth voltage conversion chip U1 is modeled as TPS54340, and the fourth voltage conversion chip U1
  • the tube drive signal reference point pin SW is connected to the adjustable voltage output terminal through the energy storage inductor L1, and the voltage divider resistors R32 and R47 connected in series to the adjustable voltage output terminal perform voltage sampling and input to the reference voltage pin of the fourth voltage conversion chip U1 FB, through the capacitors C14, C40, C42, C54, C55, and C56 connected in parallel to the adjustable voltage output terminal to filter the adjustable voltage output.
  • the upper tube drive signal reference point pin SW of the fourth voltage conversion chip U1 is also connected with freewheeling Diode D2
  • enable pin EN is also connected to resistor R33
  • resistor timing/external clock pin RT/CLK is also connected to frequency divider resistor R85
  • frequency compensation pin COMP is also connected to adjust loop and stabilize voltage output
  • the switch control signal receiving end is connected to the main control unit to receive the switch control signal
  • the resistors R104 and R105 are connected in series between the 3.3V voltage and the switch control signal receiving end
  • the emitter of the transistor Q12 is connected to 3.3V
  • the voltage and base are connected to the node between resistors R104 and R105 and the collector is connected to the enable pin EN of the fourth voltage conversion chip U1.
  • the switch control signal is L
  • the transistor Q12 is turned on and the fourth voltage conversion chip U1 is turned on jobs.
  • the circuit principle of the first PWM signal receiving circuit the first PWM signal received by the first PWM signal receiving terminal is filtered by RC filtering and then input to the non-inverting input terminal of the first voltage follower U3B, and the output terminal of the first voltage follower U3B It is connected to the node between the voltage dividing resistors R32 and R47 through the voltage dividing resistor R54, wherein the resistor R45, the capacitor C63, the resistor R40 and the capacitor C52 form an RC filter, and then input to the non-inverting input of the first voltage follower U3B through the resistor R36 A capacitor C50 is also connected to the non-inverting input terminal of the first voltage follower U3B.
  • the animal and plant tissue ablation instrument also includes an output voltage sampling unit for collecting the adjustable voltage of the DCDC power adjustment unit, and the collected adjustable voltage is sent to the main control unit through the second voltage follower U3A.
  • the voltage divider resistors R55 and R60 connected in series at the adjustable voltage output terminal are used for voltage sampling and filtered through RC filtering, and then input to the non-inverting input terminal of the second voltage follower U3A, and the second voltage follows
  • the positive voltage terminal of the U3A is connected to the 3.3V voltage
  • the output terminal of the second voltage follower U3A is connected to the main control unit.
  • the resistor R59 and the capacitor C39 form an RC filter
  • the 3.3V voltage is also connected to a capacitor C44.
  • the second voltage follower A capacitor C43 is also connected to the output of U3A.
  • the driving unit includes a first driving unit and a second driving unit, wherein the first driving unit includes a first driving MOS transistor Q6, and the second driving unit includes a second driving MOS transistor Q2.
  • the second PWM signal receiving terminal N of the first driving unit receives the first second PWM signal sent by the main control unit.
  • the output terminal of the first driving unit is connected to the end of the same name of the primary coil of the transformer unit.
  • the second PWM signal receiving terminal P receives the second second PWM signal sent by the main control unit, and the output terminal of the second driving unit is connected to the synonymous terminal of the primary coil of the transformer unit, and is received through the second PWM signal receiving terminal N
  • the first second PWM signal controls the first driving MOS transistor Q6 to turn on/off
  • the second second PWM signal received by the second PWM signal receiving terminal P controls the second driving MOS transistor Q2 to turn off/on.
  • the transformation unit is a push-pull transformer
  • the adjustable voltage output end of the DCDC power adjustment unit is connected to the primary side of the push-pull transformer.
  • the synonymous end of the first coil and the same name of the second coil The resistors R10 and R14 are connected in series between the ground and the second PWM signal receiving terminal N.
  • the gate of the first driving MOS transistor Q6 is connected to the node between the resistors R10 and R14, the source is grounded, and the drain is the first driving unit.
  • the output terminal is also connected to the end of the same name of the first coil of the primary side of the push-pull transformer; resistors R5 and R13 are connected in series between the ground and the second PWM signal receiving terminal P, and the gate of the second driving MOS transistor Q2 is connected to one of the resistors R5 and R13
  • the source is grounded, and the drain is the output end of the second driving unit and is connected to the synonymous end of the second coil of the primary side of the push-pull transformer.
  • the first driving MOS transistor Q6 When the first second PWM signal received by the second PWM signal receiving terminal N is H and the second PWM signal received by the second PWM signal receiving terminal P is L, the first driving MOS transistor Q6 is controlled to be turned on, The second driving MOS tube Q2 is turned off, the input loop of the first coil of the primary side is connected and the input current direction is from the synonymous end of the first coil of the primary side to the same name end, the input loop of the second coil of the primary side is turned off, and the secondary side The output current direction of the output loop of the coil is from the same name end to the different name end of the secondary coil.
  • the first driving MOS transistor Q6 is controlled to be turned off, and the first The second drive MOS tube Q2 is turned on, the input loop of the second coil of the primary side is connected and the input current direction is from the same name end of the second coil of the primary side to the different name end, the input loop of the first coil of the primary side is cut off, and the secondary side
  • the output current direction of the output loop of the coil is from the different-named end of the secondary coil to the same-named end.
  • the input DC voltage is converted into an AC waveform with a frequency of 30KHz and a voltage of hundreds of volts, which provides conditions for the subsequent resonant unit.
  • the resonant unit is arranged in the output loop of the push-pull transformer.
  • the resonant unit is LC series resonance and includes an inductor T1 and a capacitor C1 connected in series in the output loop of the push-pull transformer.
  • the inductor T1 uses EFD20.
  • the ultrasonic transducer is arranged in the output loop of the push-pull transformer.
  • the ultrasonic transducer is connected through the interface J2.
  • the sampling unit collects the output loop voltage and current of the push-pull transformer.
  • the sampling unit includes a voltage sampling unit and a current sampling unit.
  • the voltage sampling unit collects the output loop voltage of the push-pull transformer.
  • the output loop of the push-pull transformer is equipped with series sampling resistors R1, R2, R3, R4 and R15.
  • the voltage sampling terminal is connected to the node between the resistors R4 and R15, and
  • the collected voltage signal is sent to the voltage sampling unit for filtering and amplification, and then sent to the main control unit for resonance adjustment.
  • the collected voltage signal is filtered through RC filtering in turn, and then filtered by the capacitor C67, then input to the non-inverting input terminal of the first operational amplifier U7B, and the inverting input terminal and output of the first operational amplifier U7B
  • the negative feedback resistor R63 is connected between the terminals, the output terminal of the first operational amplifier U7B is filtered by RC filtering, and then input to the non-inverting input terminal of the first comparator U8A through the resistor R72, which is connected in series between the 3.3V voltage and the ground
  • the voltage divider resistors R77 and R76 conduct voltage sampling and input to the inverting input terminal of the first comparator U8A.
  • the output terminal of the first comparator U8A is connected to the main control unit through a resistor R71, and the resistor R72 is connected to the non-inverting input terminal of the first comparator U8A.
  • the other end is also connected to the main control unit through a resistor R70, where resistor R61 and capacitor C65 form an RC filter to filter the collected voltage signal, and resistor R62 and capacitor C66 form an RC filter to filter the signal output from the output terminal of the first operational amplifier U7B.
  • the inverting input terminal of the first operational amplifier U7B is also connected in series with a resistor R64 and a capacitor C70, and the non-inverting input terminal of the first operational amplifier U7B is also connected with a DC 3.3V voltage, which passes through the voltage divider resistors R56 and R58. After dividing the voltage with R57, a divided voltage is generated and input to the non-inverting input terminal of the first operational amplifier U7B. The divided voltage is also filtered by a capacitor C64.
  • resistor R58 is connected to the non-inverting input terminal of the first operational amplifier U7B and the other end is connected to the resistor R56 ,
  • the resistor R56 is connected to the 3.3V voltage, one end of the resistor R57 is connected to the node between the resistors R56 and R58 and the other end is grounded.
  • the positive voltage terminal of the first comparator U8A is connected to the 3.3V voltage.
  • the 3.3V voltage passes through the parallel capacitor C76 and C77 filtering.
  • the current sampling unit collects the output loop current of the push-pull transformer, and the current sampling terminal is connected in series with the output loop of the push-pull transformer, and sends the collected current signal to the current sampling unit for filtering, amplification, and then to the main control unit for resonance adjust.
  • the circuit principle of the current sampling unit is elaborated in detail: the collected current signal is filtered through RC filtering in turn, and then blocked by the capacitor C75, and then input to the non-inverting input terminal of the second operational amplifier U7A, and the inverting input terminal and output of the second operational amplifier U7A
  • the negative feedback resistor R73 is connected between the terminals, the output terminal of the second operational amplifier U7A is filtered by RC filtering, and then input to the non-inverting input terminal of the second comparator U8B through the resistor R81, which is connected in series between the 3.3V voltage and the ground
  • the voltage divider resistors R82 and R83 are input to the inverting input terminal of the second comparator U8B for voltage sampling.
  • the output terminal of the second comparator U8B is connected to the main control unit through a resistor R80, and the resistor R81 is connected to the non-inverting input terminal of the second comparator U8B.
  • the other end is also connected to the main control unit through a resistor R79, where resistor R65 and capacitor C68 form an RC filter to filter the collected current signal, and resistor R69 and capacitor C69 form an RC filter to filter the signal output from the output terminal of the second operational amplifier U7A.
  • the inverting input terminal of the second operational amplifier U7A is also connected in series with a resistor R74 and a capacitor C78, and the non-inverting input terminal of the second operational amplifier U7A is also connected with a DC 3.3V voltage, which passes through the voltage divider resistors R78 and R66. , R67 and R68 are divided to generate a divided voltage and input to the non-inverting input of the second operational amplifier U7A. The divided voltage is filtered by a capacitor C71.
  • One end of the resistor R68 is connected to the non-inverting input of the second operational amplifier U7A and the other is connected in series.
  • the main control unit outputs the first PWM signal to control the DCDC power adjustment unit to output an adjustable voltage, and through the feedback of the sampling unit that collects the output loop voltage and current of the transformer unit, outputs two second PWM signals with complementary duty cycles To the first driving MOS tube Q6 and the second driving MOS tube Q2 of the driving unit.
  • the main control unit includes a main control chip U2 and a main control chip peripheral circuit.
  • the model of the main control chip U2 is N32905U1DN, adopts an ARM9 core, and has a main frequency of 200MHz.
  • the main control chip peripheral circuit includes system clock, reset, etc.
  • N32905U1DNN3290x integrates JPEG codec, CMOS sensor interface, 32-channel SPU (sound processing unit), ADC, DAC, which can meet various application requirements while saving BOM cost.
  • the maximum resolution of N32905U1DNN3290x is XVGA(1,024x768)@TFT LCD panel.
  • the 2D BitBLT accelerator accelerates graphics calculations, smooths rendering, and offloads the CPU to save power.
  • N32905U1DNN3290x specially adopts 1Mbitx16 3.3V SDRAM design.
  • N32905U1DNN3290x specially adopts 4Mbitx16 1.8V DDR SDRAM design.
  • a 16Mbitx16 1.8V DDR2SDRAM is stacked inside the N32905U1DNN3290x to ensure higher performance and minimize system design work such as EMI and noise coupling.
  • the animal and plant tissue ablation instrument of the present invention uses the first PWM signal to control the DCDC power adjustment unit to output an adjustable voltage, so that the output power is adjustable from 0.5 to 1000W.
  • this embodiment further discloses an animal and plant tissue ablation instrument based on the principle of ultrasound.
  • the main control unit of the present invention also includes auxiliary chip U1 and auxiliary chip peripheral circuits.
  • the model is STM32F031G4U6.
  • the main control chip U2 of the main control unit sends instructions to the auxiliary chip U1 by collecting feedback from the sampling unit of the output loop voltage and current of the transformer unit.
  • the auxiliary chip U1 receives the instructions from the main control chip U2 and outputs two channels with a frequency of 30KHz.
  • the second PWM signal with complementary duty cycle is sent to the first driving MOS transistor Q6 and the second driving MOS transistor Q2 of the driving unit.
  • the two channels of frequency are 30KHz
  • the second PWM signal with complementary duty cycle is also fed back to the main control Chip U2.
  • the signals output from the output terminal of the first comparator U8A and the output terminal of the second comparator U8B are sent to the auxiliary chip U1.
  • the two second PWM signals with a frequency of 30KHz and a complementary duty cycle are amplified by the first signal amplifier output circuit and the second signal amplifier output circuit respectively, and then sent to the first driving MOS transistor Q6 and the second driving MOS of the driving unit.
  • the second PWM signal is input to the base of the first amplifying transistor Q5 through the series voltage divider resistors R49 and R53 for amplification, and the collector of the first amplifying transistor Q5 is connected to 12V through the resistor R34 Voltage, the emitter of the first amplifying transistor Q5 is grounded, the collector of the first amplifying transistor Q5 is connected to the bases of the first output transistor Q2 and the second output transistor Q4, the first output transistor Q2 is a P-type transistor, and the second output The transistor Q4 is an N-type transistor.
  • the collector of the first output transistor Q2 is connected to a 12V voltage.
  • the amplified first second PWM signal is connected from the emitter of the first output transistor Q2 to the emitter of the second output transistor Q4.
  • the second PWM signal is input to the base of the second amplifying transistor Q8 through the series voltage divider resistors R86 and R87 for amplification, and the collector of the second amplifying transistor Q8 is connected to 12V through the resistor R84 Voltage, the emitter of the second amplifying transistor Q8 is grounded, the collector of the second amplifying transistor Q8 is connected to the bases of the third output transistor Q6 and the fourth output transistor Q7, the third output transistor Q6 is a P-type transistor, and the fourth output Transistor Q7 is an N-type transistor, the collector of the third output transistor Q6 is connected to a 12V voltage, and the amplified second PWM signal is connected from the emitter of the third output transistor Q6 to the emitter of the fourth output transistor Q7.
  • the third output transistor Q6 When the second PWM signal input is H, the third output transistor Q6 is turned on, and the fourth output transistor Q7 is turned off; when the second PWM signal input is L, the third output transistor Q6 is turned off, and the fourth output transistor Q7 is turned on, Thus, the amplified square wave second channel second PWM signal is output.
  • the main control chip U2 is also connected to a display unit.
  • the display unit includes a display chip J8 and an LCD display connected to the display chip J8.
  • the model of the display chip J8 is FPC050.
  • the display chip J8 is connected to the 3.3V voltage.
  • the signal of the main control chip U2 LCD_BL is amplified by MOS tube Q1 and sent to display chip J8.
  • the main control chip U2 is also connected with buttons.
  • This embodiment includes four buttons, namely the first button, the second button, the third button, and the fourth button.
  • the first button is the left button
  • the second button is the right button
  • the second button is the right button.
  • the three buttons are the middle button
  • the fourth button is the OK button.
  • the first button includes a pull-up resistor R4 and a pull-down resistor R11
  • the pull-up resistor R4 is connected to a 3.3V voltage
  • the node between the pull-up resistor R4 and the pull-down resistor R11 is connected to one end of the button S2, and the other end of the button S2 is grounded and pulled down
  • the other end of the resistor R11 is connected to the main control chip U2.
  • the pull-down resistor R11 When the button S2 is pressed, the pull-down resistor R11 is only 1K, and the first button outputs a low level. When the button S2 is released, the output is pulled up by the pull-up resistor R4 and the pull-down resistor R11. The first button outputs a high level; the second button includes a pull-up resistor R6 and a pull-down resistor R19.
  • the pull-up resistor R6 is connected to a 3.3V voltage.
  • the node between the pull-up resistor R6 and the pull-down resistor R19 is connected to one end of the button S3, and the button S3
  • the other end of the pull-down resistor R19 is connected to the main control chip U2.
  • the second button When the button S3 is pressed, the second button outputs a low level.
  • the third button includes a pull-up resistor R8 and a pull-down resistor R20.
  • the pull-up resistor R8 is connected to a 3.3V voltage.
  • the node between the pull-up resistor R8 and the pull-down resistor R20 is connected to one end of the button S4, and the button S4
  • the other end of the pull-down resistor R20 is connected to the main control chip U2.
  • the third button outputs a high level;
  • the fourth button includes a pull-up resistor R10 and a pull-down resistor R26.
  • the pull-up resistor R10 is connected to a 3.3V voltage.
  • the node between the pull-up resistor R10 and the pull-down resistor R26 is connected to one end of the button S5, and the button S5
  • the other end of the pull-down resistor R26 is connected to the main control chip U2.
  • the main control chip U2 is also connected to a memory, and the memory uses an SPI-FLASH device to store parameters, including resonance parameters, setting parameters, and so on.
  • the main control chip U2 is also connected with a USB interface unit.
  • the USB interface unit includes a USB chip ESD1 connected to the main control chip U2 and an interface J7 connected to the USB chip ESD1.
  • the model of the USB chip ESD1 is USBLC6, and the model of the interface J7 is SIP254.
  • the main control chip U2 is also connected to a touch unit, which includes voltage divider resistors R99 and R101 connected in series between the 3.3V voltage and ground, a resistor R100 connected to the node between the resistors R99 and R101, and one of the base connection resistors R99 and R101.
  • Transistor Q10 at the node between the transistor Q10 and the 3.3V voltage is connected in series with voltage divider resistors R98 and R96.
  • the node between the voltage divider resistors R98 and R96 is connected to the gate of the MOS transistor Q9 and the source of the MOS transistor Q9.
  • the drain of the MOS transistor Q9 is connected to the touch interface J1, the touch interface J1 is connected to the main control chip U2, the touch interface J1 is connected to the touch panel, and the power enable signal TP_PWEN sent by the main control chip U2 to the triode through the resistor R100
  • the base of Q10 when the power enable signal TP_PWEN is H, the transistor Q10 is turned on, and the MOS transistor Q9 is turned on.
  • the main control chip U2 is also connected with an external interface J4.
  • the main control chip U2 is also connected with a buzzer.
  • This embodiment provides an extraction solution, which includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride, and the pH of the extraction solution is adjusted to 4 by acetic acid.
  • the extract is applied to preserve tissues or cells.
  • This embodiment provides an extraction liquid, including:
  • the extract is adjusted to pH 4 with acetic acid.
  • the extract is used in RNA extraction.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B;
  • the extract A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin and 0.01mol/L SDS; the remainder is solvent.
  • the pH of the extract A and the extract B are both 4.
  • the amount of RNA extracted using the extraction solution in this embodiment is 1000-1800 ⁇ g/mL, and it is measured that A260/A280 is in the range of 1.6-1.9, and A260/A230 is in the range of 1.6-1.9.
  • the picture of agarose gel electrophoresis is shown in Figure 7.
  • the method for rapidly extracting RNA using the above-mentioned extracting solution A and extracting solution B and using the animal and plant tissue ablation instrument based on the ultrasonic principle in Example 1 or Example 2 includes the steps: 1. Firstly, using the extracting solution A to store the tissue or cells, the The extraction solution A includes 1.2-2.0mol/L guanidine thiocyanate and 0.5-1.5mol/L guanidine hydrochloride, and the pH is 4;
  • extract B to the tissues or cells stored in extract A, and mix upside down to obtain a mixture.
  • the extract B includes 30-65vt% water-saturated phenol and 9-15vt% in volume fraction Glycerin, the balance is solvent;
  • Extraction solution B also includes 0.20-1mol/L guanidine thiocyanate, 0.4-0.8mol/L ammonium thiocyanate and 0.0073-0.018mol/L surfactant in molar concentration, and the pH is adjusted to 4;
  • RNA from tissues use the mixture obtained in step 2 to ultrasonically disrupt the animal and plant tissue ablation instrument based on the principle of ultrasound; when extracting RNA from cells, proceed directly to the operation in step 5 below;
  • the use of the A solution of the present invention this time increases and prolongs the tissue preservation time.
  • the A solution of the present invention is used to compare the preservation effect with the RNA extraction reagent Trizol on the market.
  • the above two reagents are used to treat rat spleen tissues. Save it at room temperature and 37°C.
  • the storage time is set at more than 7 days. After the preservation, the tissue morphology is observed. It is found that all the spleen tissues stored in Trizol have been dissolved.
  • the liquid A invented by our side is normal.
  • RNA extraction was performed on the two kinds of preserved spleen tissues. As a result, the amount of RNA extracted from the solution A of this example was more than 3 times higher than that of the Trizol preserved tissue.
  • the present invention is compared with the extraction results of the spleen tissue preserved by Trizol, and the result is that the amount of RNA extracted from the tissue after the preservation of Trizol is 474 ⁇ g/mL, and the measured A260/A280 is around 1.583, and the A260/A230 is around 2.563.
  • the amount of RNA extracted using the RNAlater of the present invention is 1241 ⁇ g/mL, and it is measured that A260/A280 is around 1.8 and A260/A230 is around 2.2.
  • the experiment 1 data is obtained and the results are analyzed.
  • the results obtained can prove that the extraction solution B can obtain higher quality RNA extraction purity than the existing TRIzol solution.
  • the RNA extracted after mixing extraction solution A and extraction solution B yields better quality RNA than the existing Trizol solution.
  • the above two reagents are used to extract RNA from rat spleen tissue, and the extracted RNA
  • the amount of RNA extracted by Trizol is generally 400 ⁇ g/mL, while the A260/A230 is determined, and the measured value is generally at the level of 1.5.
  • the A260/A280 measurement result is at the 1.8 level.
  • the amount of RNA extracted by the reagent of this embodiment is above 1200 ⁇ g/mL, while the A260/A230 is measured, and the measured value is generally at the 1.8 level.
  • the A260/A280 measurement result is at the 1.7 level.
  • the extracted RNA has high extraction purity.
  • the RNA extracted by our reagent is confirmed by 1% agarose gel electrophoresis.
  • the RNA extracted by the reagent of this example The quality and effect are better than the RNA extracted by trizol.
  • the result of the comparison between the two is that the amount of RNA extracted by Trizol is generally 400 ⁇ g/mL, while the A260/A230 is measured, and the measured value is generally at the level of 1.5.
  • the A260/A280 measurement result is at the 1.8 level.
  • the amount of RNA extracted by the reagent of this embodiment is above 1200 ⁇ g/mL, while the A260/A230 is measured, and the measured value is generally at the 1.8 level.
  • the A260/A280 measurement result is at the 1.7 level.
  • RNA extraction and quality and purity evaluation were performed.
  • the amount of RNA extracted in this example was above 1200 ⁇ g/mL.
  • the A260/A230 are measured, and the measured value is generally at the 1.8 level.
  • the A260/A280 measurement result is at the 1.7 level.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B;
  • the extract A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% ⁇ - Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt ⁇ bromophenol blue, 0.15mol/L sodium acetate, 8vt% isopropanol, 0.1wt% 8-hydroxyquinoline and 0.1mmol/L EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • the experiment is carried out in a controlled variable manner by setting up multiple comparison groups.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerol, 0.3wt% SDS, 0.1wt% ⁇ -mercaptoethanol, 0.08mmol/L citric acid Potassium, 0.1wt% of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, 0.1wt% of 8-hydroxyquinoline, and 0.1mmol/L of EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerol, 0.1wt% ⁇ -mercaptoethanol, 0.08mmol/L L potassium citrate, 0.1 wt ⁇ bromophenol blue, 0.15 mol/L sodium acetate, 8 vt% isopropanol, 0.1 wt% 8-hydroxyquinoline and 0.1 mmol/L EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% ⁇ - Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt ⁇ bromophenol blue, 0.15mol/L sodium acetate, 8vt% isopropanol, 0.1wt% 8-hydroxyquinoline and 0.1mmol/L EDTA.
  • the pH of the extraction liquid A and the extraction liquid B are both 7 or 2.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.08mmol/L lemon Potassium acid, 0.1wt% of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, 0.1wt% of 8-hydroxyquinoline and 0.1mmol/L of EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% ⁇ - Mercaptoethanol, 0.1wt ⁇ of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, 0.1wt% of 8-hydroxyquinoline, and 0.1mmol/L of EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.08mmol/L lemon Potassium acid, 0.1wt ⁇ of bromophenol blue, 0.15mol/L of sodium acetate, 8vt% of isopropanol, and 0.1mmol/L of EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% ⁇ - Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt ⁇ bromophenol blue, 8vt% isopropanol, 0.1wt% 8-hydroxyquinoline, and 0.1mmol/L EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% ⁇ - Mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt ⁇ bromophenol blue, 0.15mol/L sodium acetate, 8vt% isopropanol, and 0.1wt% 8-hydroxyquinoline.
  • the pH of extract A and extract B was adjusted to 4 with acetic acid.
  • This embodiment provides an extraction solution, including extraction solution A and extraction solution B; wherein extraction solution A includes 1.6 mol/L guanidine thiocyanate and 1 mol/L guanidine hydrochloride;
  • the extract B includes 57vt% water-saturated phenol, 1mmol/L DTT, 0.24mol/L guanidine thiocyanate, 0.6mol/L ammonium thiocyanate, 11.25vt% glycerin, 0.3wt% SDS, 0.1wt% ⁇ -mercaptoethanol, 0.08mmol/L potassium citrate, 0.1wt ⁇ bromophenol blue, 5mol/L urea, 0.15mol/L sodium acetate, 8vt% isopropanol, 0.1wt% Of 8-hydroxyquinoline and 0.1mmol/L EDTA.
  • the pH of the extract A and the extract B were adjusted to 4 with acetic acid.
  • the results are analyzed as follows: The lack of guanidine thiocyanate in experimental group 1 directly caused insufficient lysis, which affected the constant of RNA extraction. The amount of RNA extracted is significantly reduced. Compared with the extraction results of the original formula, the amount of RNA extracted by the lack of 0.24mol/L guanidine thiocyanate reagent is less, and the amount of RNA extracted will be around 300 ⁇ g/mL, and A260/A230 1.7 Up and down, A260/A280 is above 2, use a spectrophotometer for quantitative determination, and use agarose gel electrophoresis to determine the purity of the extracted RNA. In experimental group 2, the lack of 0.3wt% SDS directly led to insufficient lysis, which affected the constant of RNA extraction.
  • RNA extracted is significantly reduced. Compared with the original extraction results, the amount of RNA extracted from the lack of 0.3wt% SDS reagent is too small. SDS directly affects the lysis. Quantitative determination is performed by spectrophotometer, and agarose gel electrophoresis is used for quantitative determination. The purity of the extracted RNA was judged, and the yield of RNA was around 200 ⁇ g/mL.
  • the experimental group 5 lacked 0.08mmol/L potassium citrate, which would affect the reagent buffer system and thus affect the experimental results.
  • the experimental group 6 lacks 8-hydroxyquinoline, because 8-hydroxyquinoline functions to maintain the stability of water-saturated phenols and prevent the oxidation of water-saturated phenols. Affect the long-term storage and stability of B liquid.
  • the increase in sugar pollution affects the quality of RNA extraction.
  • the lack of EDTA in experimental group 8 will increase the risk of RNA being degraded by RNase. Because 5mol/L urea and 1mmol/L DTT both inhibit the effect of RNase, so that the extracted RNA is protected from degradation. Therefore, the RNA stored in experimental group 9 has been degraded.
  • the present invention is not limited to the above-mentioned alternative embodiments.
  • anyone can derive other products in various forms under the enlightenment of the present invention, but regardless of any changes in its shape or structure, all that fall into the scope of the claims of the present invention The technical solutions within the scope fall within the protection scope of the present invention.

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Abstract

利用基于超声原理的动植物组织消融仪快速提取RNA的方法，1)先用提取液A保存的组织或细胞；再向提取液A保存的组织或细胞中加入提取液B，混匀获得混合物；2)从组织中提取RNA时，将混合物进行超声破碎；3)从细胞中提取RNA时，直接进行下述步骤5)中的操作；4)将超声破碎后的混合物颠倒混匀；5)在步骤3)从细胞提取RNA的混合物或步骤4)的混合物中，加入氯仿离心，取上层液体；6)在上层液体中加入异丙醇再离心获得沉淀物；7)向沉淀物中加入乙醇溶液，混匀后离心，弃去液体；8)最后加入水溶解。

Description

利用基于超声原理的动植物组织消融仪快速提取RNA的方法 技术领域
本发明属于生物制剂技术领域，具体涉及利用基于超声原理的动植物组织消融仪快速提取RNA的方法。
背景技术
现代分子生物学实验、临床分子诊断中常常要从组织和细胞中分离和纯化RNA，而RNA的质量高低常常会影响cDNA库、RT-PCR和Northern Blot等分子生物学实验的成败。
现在常用的方法是先通过组织保护液对组织进行保护，再采用提取液对组织或细胞中的RNA进行提取。常用的保护方式包括液氮保护和保护液保护，所述常用保护液包括RNA later试剂和RNA safety试剂。
其中，液氮保护方式不仅所需条件苛刻，且成本较高，仅适合临时性的组织保护。而保护液通常也需要在低温下进行贮藏，且其中RNA later溶液与TRIzol、硅胶柱和磁珠法的化学成分不兼容，RNA later溶液中含有异硫氰酸铵，当组织块从RNA later溶液中取出用于提取RNA时，若未将组织块上带有的RNA later溶液清洗干净，则RNA later溶液中的异硫氰酸铵会与提取液中的酚、SDS结合或反应生成沉淀，导致提取液成分和浓度改变，裂解不彻底，降低RNA的纯度和收率。因此，采用RNA later溶液保存的组织或细胞在提取前通常需要进行反复地清洗，浪费时间和人力，降低提取效率。
而提取RNA则常常采用TRIzol法、硅胶柱和磁珠法等，但这些方法普遍存在RNA收率低、杂质污染严重的问题，不能满足后续基因诊断、生物芯片分析、基因表达分析等实验的质量要求。
此外，组织块的破碎过程也极大的影响着RNA的质量。常用组织块的破碎方法包括液氮研磨法、匀浆法(手工匀浆、机器匀浆、超声匀浆)和反复冻融法。液氮研磨法需要的组织块较大，且较难将组织块彻底打碎；匀浆法较难彻底抑制内源性RNA酶活性，容易造成内源性降解；反复冻融法的缺点也是难以彻底裂解大块组织，且内源性RNA酶活性难以抑制。因此，对动物源性组织块的RNA提取存在操作复杂、破碎不完全、内源性RNA活性难于抑制等困难，造成RNA的提取质量及产量难以保障。
发明内容
为了解决现有技术存在的上述问题，本发明目的在于提供利用基于超声原理的动植物组织消融仪快速提取RNA的方法。
本发明所采用的技术方案为：
利用基于超声原理的动植物组织消融仪快速提取RNA的方法，包括步骤：
G1.先用提取液A保存组织或细胞，所述提取液A包括1.2-2.0mol/L的硫氰酸胍和0.5-1.5mol/L的盐酸胍，且pH为4；
G2.再向提取液A保存的组织或细胞中加入提取液B，颠倒混匀，获得混合物，所述提取液B包括以体积分数计的30-65vt％的水饱和苯酚和9-15vt％的甘油，余量为溶剂；
提取液B还包括以摩尔浓度计的0.20-1mol/L的硫氰酸胍、0.4-0.8mol/L的硫氰酸铵和0.0073-0.018mol/L的表面活性剂，且pH调节为4；
G3.从组织中提取RNA时，将步骤G2得到的混合物利用基于超声原理的动植物组织消融仪进行超声破碎；从细胞中提取RNA时，直接进行下述步骤G5中的操作；
G4.将超声破碎后的混合物颠倒混匀；
G5.在步骤G3从细胞提取RNA的混合物或步骤G4的混合物中，加入氯仿，混匀后离心，取上层液体；
G6.在上层液体中加入异丙醇，混匀后离心，弃去液体，获得沉淀物；
G7.向沉淀物中加入乙醇溶液，混匀后离心，弃去液体；
G8.待乙醇挥发后加入水溶解；
所述基于超声原理的动植物组织消融仪，包括：电源、DCDC转换单元、主控单元、DCDC功率调节单元、驱动单元、变压单元、谐振单元、超声换能器和采样单元；
所述电源，通过DCDC转换单元和DCDC功率调节单元转换电压后给动植物组织消融仪使用；主控单元，输出第一PWM信号控制DCDC功率调节单元输出可调电压，并通过采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元并经过变压单元变压，然后通过设于变压单元的输出端回路中的谐振单元调节谐振点到超声振子的共振点，使得设于变压单元的输出端回路中的超声振子工作在谐振状态。
超声振子包括超声换能器和将超声波换能器输入的振动改变振幅后传递出去的变幅杆。
在上述技术方案的基础上，所述提取液B中的表面活性剂为十二烷基硫酸钠。
在上述技术方案的基础上，所述提取液A采用盐酸调节pH值，所述提取液B采用乙酸调节pH值。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为6.4mmol/L-19.20mmol/L的β-巯基乙醇。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.05-0.15mmol/L的柠檬酸钾。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为74.62μmol/L-223.87μmol/L的溴酚蓝。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.01-2mol/L的乙酸钠。
在上述技术方案的基础上，所述提取液B中还包括体积分数为5-25％的异丙醇。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.689mmol/L-68.9mmol/L的8-羟基喹啉。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.5-1000mmol/L的DTT。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.5-5mol/L的尿素。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.1-500mmol/L的EDTA。
在上述技术方案的基础上，所述提取液B中还包括摩尔浓度为0.01-10mmol/L的乙酸铵。
在上述技术方案的基础上，所述步骤G8中的水为双蒸水、去离子水或超纯水。
在上述技术方案的基础上，所述提取液A和提取液B的总质量为组织或细胞质量的100-150倍。
在上述技术方案的基础上，所述步骤G3中，超声功率为0.5-1000W，超声时间为0.5-30s。
在上述技术方案的基础上，所述步骤G5中，氯仿的体积为混合物体积的0.1-0.4倍。
在上述技术方案的基础上，所述步骤G6中，异丙醇与上层液体的体积比为1：1。
首先，本发明的提取液主要成分为硫氰酸胍和盐酸胍，所述硫氰酸胍是一种有机化合物，分子式为C 2H 6N 4S，主要用于生物医药，化学试剂等。
同时硫氰酸胍为解偶剂、强力蛋白质变性剂、破坏蛋白质二级结构；主要起细胞裂解作用，促进核蛋白和核酸分离；以及起着酶抑制剂作用，具有保存组织效果。
而所述的盐酸胍为一种白色或微黄色块状物，作为提取细胞总RNA中的强烈变性剂。盐酸胍溶液可溶解蛋白质，导致细胞结构破坏，核蛋白二级结构破坏，从核酸上解离下来，此外，RNA酶可被盐酸胍等还原剂灭活，作为核酸酶的抑制剂，能够从富含RNase组织中提取RNA。也就是说，硫氰酸胍能够裂解组织或细胞，但同时通过增加的盐酸胍能够抑制RNA酶的活性，从而达到裂解并保存的效果。
通过硫氰酸胍和盐酸胍的定量配置从而对组织或细胞提供一定时间的常温保存效果，一般可在常温下保存超过7天。
可在提取液中采用磷酸二氢钠、氯化钠和氯化镁作为缓冲体系，从而保持盐离子浓度。而其中包含的水饱和酚具有裂解细胞和沉淀蛋白的作用，处于酸性时有助去除DNA；而硫氰酸铵为无色结晶，易潮解、易溶于水和乙醇，溶于甲醇和丙酮，几乎不溶于氯仿和乙酸乙酯，其能够沉淀蛋白质，从而达到分离杂质的效果；所述的表面活性剂采用SDS，即十二烷基硫酸钠，具有使蛋白变性的作用；而通过调节pH值至3.5-4.5主要是起到保证提取质量的效果，因为pH过低主要会降低提取的纯度，而过高则会影响产量。
增加的β-巯基乙醇，它兼具乙二醇和乙二硫醇的官能团，为挥发性液体，具有较强烈的刺激性气味。βME通常用于二硫键的还原，可以作为生物学实验中的抗氧化剂。它被广泛使用的原因是它的羟基使它能够溶解于水中，并且降低它的挥发性。在本发明中主要起到破坏RNase中的二硫键，从而起到抑制效果。
增加的柠檬酸钾是一种常用的缓冲剂，它是一种白色，略带吸湿性结晶性粉末。无臭，有生理盐水的味道，易溶于水，缓溶于甘油，不溶于醇，味咸而凉。作稳定剂和pH缓冲剂等，参与缓冲体系，作为酶的抑制剂。
增加的溴酚蓝的分子式为C 19H 10Br 4O 5S，为浅黄色到棕黄色粉末，通常作为电泳指示染料，而在本发明的配方中也同样作为颜色指示剂。在离心过程中，溴酚蓝随DNA和蛋白质下沉，可用于指示RNA离析是否完成。
增加的乙酸钠能够在RNA提取过程中沉淀溶液中的糖类，从而达到去掉杂质的效果，增加提取的RNA的纯度。
增加的异丙醇为一种有机化合物，为无色透明液体，通过-OH的疏水作用使得RNA链中的亲水基团得到保护，同时具有还原上层液RNA的作用。
增加的8-羟基喹啉同样可以抑制RNase，与氯仿联合使用可增强抑制作用。
增加的DTT为一种还原剂，与巯基乙醇的作用一致，主要起到破坏RNase中的二硫键，从而起到抑制效果。
增加的尿素是一种白色晶体状的有机化合物；高浓度尿素可以是蛋白质变性，并同样可以抑制RNase活性。
增加的EDTA是一种二价金属螯合物，同样作为酶的抑制剂。
增加的乙酸铵结构简式为CH 3COONH 4，又称醋酸铵。是一种有乙酸气味的白色三角晶体，可作为分析试剂和肉类防腐剂。在本发明中沉淀RNA提取中遇到的蛋白质，但铵根离子容易与硫代硫酸根离子形成白色沉淀。
其中，提取液A可以室温保存组织24小时，保证RNA不降解，即不用低温保存，脱离冷链。
本发明采用超声破碎组织，速率快，不仅避免了组织破碎不完全、内源性RNA酶的干扰，而且大大缩短提取时间，进一步提高了RNA的提取效率。
值得说明的是，其中以摩尔浓度计算的均为粉末状成分，故剩余液体组分以体积分数计算，而粉末状组分因溶解于溶剂中故其体积变化量可忽略不计。
利用基于超声原理的动植物组织消融仪快速提取RNA的方法，所述基于超声原理的动植物组织消融仪的DCDC功率调节单元包括：开关控制信号接收端连接主控单元接收开关控制信号的开关控制信号接收电路；
第一PWM信号接收端连接主控单元接收第一PWM信号的第一PWM信号接收电路；
通过主控单元发送的开关控制信号和第一PWM信号将电源转换为可调电压输出的第四DCDC转换单元。
作为优选，所述动植物组织消融仪的第四DCDC转换单元包括：依次处理电源电压的第四电压输入滤波、第四电压转换芯片U1和第四电压输出滤波，第四电压输入滤波包括连接在电源端的滤波电容，第四电压转换芯片U1的上管驱动信号参考点管脚SW通过储能电感L1连接到可调电压输出端，串联在可调电压输出端的分压电阻R32和R47进行电压采样输入到第四电压转换芯片U1的参考电压管脚FB，第四电压转换芯片U1的上管驱动信号参考点管脚SW还连接有续流二极管D2，使能管脚EN还连接有电阻R33，电阻器时序/外部时钟管脚RT/CLK还连接有频率分压电阻R85，频率补偿管脚COMP还连接有用于调节环路、稳定电压输出的电容C51、C49和电阻R46，其中，电容C49与串联的电容C51和电阻R46并联，第四电压输出滤波包括连接在可调电压输出端的滤波电容；
开关控制信号接收电路包括：串联于转换后电压与开关控制信号接收端之间的电阻R104和R105，发射极连接转换后电压、基极连接电阻R104和R105之间的节点且集电极连接第四电压转换芯片U1的使能管脚EN的三极管Q12，当开关控制信号为L时，三极管Q12导通，第四电压转换芯片U1开启工作；
第一PWM信号接收电路包括：依次处理第一PWM信号的RC滤波和第一电压跟随器U3B，第一PWM信号接收端接收的第一PWM信号通过RC滤波进行滤波后输入到第一电压跟随器U3B的同相输入端，第一电压跟随器U3B的输出端通过分压电阻R54连接到分压电阻R32和 R47之间的节点。
作为优选，所述动植物组织消融仪还包括用于采集DCDC功率调节单元的可调电压的输出电压采样单元，采集的可调电压通过第二电压跟随器U3A发送至主控单元。
作为优选，所述动植物组织消融仪的驱动单元包括第一驱动单元和第二驱动单元，第一驱动单元的第二PWM信号接收端N接收主控单元发送的第一路第二PWM信号，第一驱动单元的输出端连接变压单元的原边线圈的同名端，第二驱动单元的第二PWM信号接收端P接收主控单元发送的第二路第二PWM信号，第二驱动单元的输出端连接变压单元的原边线圈的异名端，通过第二PWM信号接收端N接收的第一路第二PWM信号控制第一驱动MOS管Q6导通/截止，通过第二PWM信号接收端P接收的第二路第二PWM信号控制第二驱动MOS管Q2截止/导通。
作为优选，所述动植物组织消融仪的变压单元为推挽变压器，DCDC功率调节单元的可调电压输出端连接推挽变压器的原边第一线圈的异名端和第二线圈的同名端；
所述第一驱动单元包括串联于地与第二PWM信号接收端N之间的电阻R10和R14，栅极连接电阻R10和R14之间的节点、源极接地且漏极为第一驱动单元的输出端并连接推挽变压器的原边第一线圈的同名端的第一驱动MOS管Q6；
所述第二驱动单元包括串联于地与第二PWM信号接收端P之间的电阻R5和R13，栅极连接电阻R5和R13之间的节点、源极接地且漏极为第二驱动单元的输出端并连接推挽变压器的原边第二线圈的异名端的第二驱动MOS管Q2。
当第二PWM信号接收端N接收的第一路第二PWM信号为H且第二PWM信号接收端P接收的第二路第二PWM信号为L时，控制第一驱动MOS管Q6导通，第二驱动MOS管Q2截止，原边第一线圈的输入端回路接通并且输入电流方向为原边第一线圈的异名端至同名端，原边第二线圈的输入端回路截止，副边线圈的输出端回路的输出电流方向为副边线圈的同名端至异名端。
当第二PWM信号接收端N接收的第一路第二PWM信号为L且第二PWM信号接收端P接收的第二路第二PWM信号为H时，控制第一驱动MOS管Q6截止，第二驱动MOS管Q2导通，原边第二线圈的输入端回路接通并且输入电流方向为原边第二线圈的同名端至异名端，原边第一线圈的输入端回路截止，副边线圈的输出端回路的输出电流方向为副边线圈的异名端至同名端，将输入的直流电压转换成频率30KHz、电压上百伏特的交流波形，为后级谐振单元提供条件。
作为优选，所述动植物组织消融仪的谐振单元为LC串联谐振，LC串联谐振包括串联于变压单元的输出端回路中的电感T1和电容C1。
作为优选，所述动植物组织消融仪的超声换能器通过接口J2设于变压单元的输出端回路中。
作为优选，所述动植物组织消融仪的采样单元包括采集变压单元的输出端回路电压的电压采样单元和采集变压单元的输出端回路电流的电流采样单元，相应地，变压单元的输出端回路设有多个串联的采样电阻；通过连接在多个采样电阻之间节点的电压采样端采集电压信号然后发送至电压采样单元进行滤波和放大，然后发送至主控单元做谐振调节；通过串联在变压单元的输出端回路的电流采样端采集电流信号发送至电流采样单元进行滤波和放大，然后发送至主控单元做谐振调节。
作为优选，所述动植物组织消融仪的主控单元包括主控芯片U2和主控芯片外围电路，主控芯片U2输出第一PWM信号控制DCDC功率调节单元输出可调电压，并通过采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元。
作为优选，所述主控单元还包括辅助芯片U1和辅助芯片外围电路，主控单元的主控芯片U2通过采集变压单元的输出端回路电压和电流的采样单元的反馈，向辅助芯片U1发送指令，辅助芯片U1接收主控芯片U2的指令，输出两路占空比互补的第二PWM信号至驱动单元，同时，两路占空比互补的第二PWM信号还反馈至主控芯片U2。
作为优选，所述两路占空比互补的第二PWM信号还分别通过第一信号放大输出电路和第二信号放大输出电路进行放大后发送至驱动单元。
作为优选，所述动植物组织消融仪还包括与主控芯片U2连接的显示单元，显示单元包括显示芯片J8和与显示芯片J8连接的LCD显示屏。
作为优选，所述动植物组织消融仪还包括与主控芯片U2连接的按键。
作为优选，所述动植物组织消融仪还包括与主控芯片U2连接的存储器。
作为优选，所述动植物组织消融仪还包括与主控芯片U2连接的USB接口单元，USB接口单元包括与主控芯片U2连接的USB芯片ESD1和与USB芯片ESD1连接的接口J7。
作为优选，所述动植物组织消融仪还包括与主控芯片U2连接的触摸单元。
本发明的有益效果为：
(1)本发明的提取液A可取代传统RNA later溶液，用于保存组织或细胞，因此，当采用提取液A和提取液B提取RNA时，不需要对组织或细胞进行清洗，省去了使用RNA later溶液保存时的清洗操作，简化了操作步骤，显著提高提取效率；
(2)本发明提取液中的各组分配比设计合理，相互之间协同作用，使得提取的RNA纯度高、收率高，且蛋白污染低；
(3)本发明的动植物组织消融仪，利用第一PWM信号控制DCDC功率调节单元输出可调电压，使功率在0.5～1000W可调，利用采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元，使谐振超声频率为20-200KHz可调，调节谐振点到超声振子的共振点，使得超声振子工作在谐振状态，输出能量最大、振幅最强，采用超声破碎组织，速率快，不仅避免了组织破碎不完全、内源性RNA酶的干扰，而且大大缩短提取时间，进一步提高了RNA的提取效率；
(4)本发明提取液A、提取液B性质均较稳定，不易受温湿度等条件影响，并能充分配合使用，可实现实验室快速、低廉、高质量、规模化提取动物RNA的目的；
(5)本发明新增盐酸胍，具有核酸酶抑制剂作用，可有效从富含RNase组织中提取RNA，新增乙酸钠具有沉淀RNA提取中遇到的糖类作用，保证提取的RNA纯度，新增异丙醇：通过-OH的疏水作用使得RNA链中的亲水基团得到保护，同时具有还原上层液RNA的作用；
(6)本发明调整了A液保存组织试剂量，可将组织用于室温保存至10天及以上，却针对于富含RNase的肝脏组织，提取得到的RNA结果正常，且产量均有提高；另外主要在RNase污染方面增加多组成分，防止RNA不被降解。
附图说明
图1是本发明-实施例的系统框图。
图2是本发明-实施例控制板的电源、DCDC转换单元和DCDC功率调节单元的电路原理图。
图3是本发明-实施例控制板的主控单元的电路原理图。
图4是本发明-实施例控制板的采样单元、显示单元和辅助芯片的电路原理图。
图5是本发明-实施例控制板的接口的电路原理图。
图6是本发明-实施例驱动板的驱动单元、变压单元和谐振单元的电路原理图。
图7是本发明实施例5中的琼脂糖凝胶电泳图片。
具体实施方式
下面结合附图及具体实施例对本发明作进一步阐述。
实施例1：
如图1-6所示，本实施例的基于超声原理的动植物组织消融仪，包括电源、DCDC转换单元、主控单元、DCDC功率调节单元、驱动单元、变压单元、谐振单元、超声换能器和采样单元。
电源，通过DCDC转换单元和DCDC功率调节单元转换电压后给动植物组织消融仪使用；主控单元，输出第一PWM信号控制DCDC功率调节单元输出可调电压，并通过采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元并经过变压单元变压，然后通过设于变压单元的输出端回路中的谐振单元调节谐振点到超声振子的共振点，使得设于变压单元的输出端回路中的超声振子工作在谐振状态。
对动植物组织消融仪做进一步的阐述。
电源为DC24V电源，DCDC转换单元包括第一DCDC转换单元、第二DCDC转换单元和第三DCDC转换单元。
其中，第一DCDC转换单元包括TVS保护、第一电压输入滤波、第一电压转换芯片和第一电压输出滤波，DC24V电源依次通过TVS保护、第一电压输入滤波、第一电压转换芯片和第一电压输出滤波转换为12V输出电压，供给主控单元和驱动单元使用。
详细阐述第一DCDC转换单元的电路原理：DC24V电源依次经过保护二极管TVS1进行TVS保护，经过三个并联在DC24V电源端的电容C59、C60和C57进行电压输入滤波，然后经过串联在DC24V电源端的分压电阻R48和R51进行电压采样输入第一电压转换芯片U6的使能管脚EN，第一电压转换芯片U6的型号为TPS54340，第一电压转换芯片U6的上管驱动信号参考点管脚SW通过储能电感L2连接到12V输出电压端，串联在12V输出电压端的分压电阻R88和R89进行电压采样输入到第一电压转换芯片U6的参考电压管脚FB，经过连接在12V输出电压端的电容C72对电压输出滤波，第一电压转换芯片U6的上管驱动信号参考点管脚SW还连接有续流二极管D4，使能管脚EN还连接有软启动电容C79，电阻器时序/外部时钟管脚RT/CLK还连接有频率分压电阻R50，频率补偿管脚COMP还连接有用于调节环路、稳定电压输出的电容C61、C62和电阻R52，其中，电容C62与串联的电容C61和电阻R52并联。
第二DCDC转换单元包括第二电压输入滤波、第二电压转换芯片和第二电压输出滤波，12V输入电压依次通过第二电压输入滤波、第二电压转换芯片和第二电压输出滤波转换 为1.8V输出电压，供给主控单元使用。
详细阐述第二DCDC转换单元的电路原理：12V输入电压依次经过两个并联在12V输入电压端的电容C84和C85进行电压输入滤波，第二电压转换芯片U9的型号为TLV62130ARGTR，第二电压转换芯片U9的MOS管驱动信号参考点管脚SW通过储能电感L3连接到1.8V输出电压端，串联在1.8V输出电压端的分压电阻R90和R91进行电压采样输入到第二电压转换芯片U9的参考电压管脚FB，经过并联在1.8V输出电压端的电容C80和C83对电压输出滤波，第二电压转换芯片U9的内部功率电源管脚PVIN、内部控制电路供电管脚AVIN、使能管脚EN还连接12V输入电压端，软启动/跟踪管脚SS/TR还连接有软启动电容C86，调节输出管脚DEF和频率配置管脚FSW均接地，输出电压采集管脚VOS与1.8V输出电压端连接。
第三DCDC转换单元包括第三电压输入滤波、第三电压转换芯片和第三电压输出滤波，12V输入电压依次通过第三电压输入滤波、第三电压转换芯片和第三电压输出滤波转换为3.3V输出电压，供给主控单元和采样单元使用。
详细阐述第三DCDC转换单元的电路原理：12V输入电压依次经过两个并联在12V输入电压端的电容C89和C90进行电压输入滤波，第三电压转换芯片U10的型号为TLV62130ARGTR，第三电压转换芯片U10的MOS管驱动信号参考点管脚SW通过储能电感L4连接到3.3V输出电压端，串联在3.3V输出电压端的分压电阻R92和R94进行电压采样输入到第三电压转换芯片U10的参考电压管脚FB，经过并联在3.3V输出电压端的电容C87和C88对电压输出滤波，第三电压转换芯片U10的内部功率电源管脚PVIN和内部控制电路供电管脚AVIN还连接12V输入电压端，经过串联在12V输入电压端的分压电阻R93和R102进行电压采样输入第三电压转换芯片U10的使能管脚EN，第三电压转换芯片U10的使能管脚EN还连接到第二电压转换芯片U9的输出正常指示信号管脚PG，当第二DCDC转换单元不正常工作时，第三DCDC转换单元则停止工作，软启动/跟踪管脚SS/TR还连接有软启动电容C91，调节输出管脚DEF和频率配置管脚FSW均接地，输出电压采集管脚VOS与3.3V输出电压端连接。
DCDC功率调节单元包括开关控制信号接收电路、第一PWM信号接收电路和第四DCDC转换单元，第四DCDC转换单元包括第四电压输入滤波、第四电压转换芯片和第四电压输出滤波，第四DCDC转换单元通过主控单元发送的开关控制信号和第一PWM信号将DC24V电源转换为0-24V可调电压输出。
详细阐述DCDC功率调节单元的电路原理：
第四DCDC转换单元的电路原理：DC24V电源依次经过三个并联在DC24V电源端的电容C6、C7和C41进行电压输入滤波，第四电压转换芯片U1的型号为TPS54340，第四电压转换芯片U1的上管驱动信号参考点管脚SW通过储能电感L1连接到可调电压输出端，串联在可调电压输出端的分压电阻R32和R47进行电压采样输入到第四电压转换芯片U1的参考电压管脚FB，经过并联在可调电压输出端的电容C14、C40、C42、C54、C55和C56对可调电压输出滤波，第四电压转换芯片U1的上管驱动信号参考点管脚SW还连接有续流二极管D2，使能管脚EN还连接有电阻R33，电阻器时序/外部时钟管脚RT/CLK还连接有频率分压电阻R85，频率补偿管脚COMP还连接有用于调节环路、稳定电压输出的电容C51、C49和电阻R46，其中，电容C49与串联的电容C51和电阻R46并联。
开关控制信号接收电路的电路原理：开关控制信号接收端连接主控单元接收开关控制信号，电阻R104和R105串联于3.3V电压与开关控制信号接收端之间，三极管Q12的发射 极连接的3.3V电压、基极连接电阻R104和R105之间的节点且集电极连接第四电压转换芯片U1的使能管脚EN，当开关控制信号为L时，三极管Q12导通，第四电压转换芯片U1开启工作。
第一PWM信号接收电路的电路原理：第一PWM信号接收端接收的第一PWM信号通过RC滤波进行滤波后输入到第一电压跟随器U3B的同相输入端，第一电压跟随器U3B的输出端通过分压电阻R54连接到分压电阻R32和R47之间的节点，其中，电阻R45、电容C63、电阻R40和电容C52组成RC滤波，然后通过电阻R36输入到第一电压跟随器U3B的同相输入端，第一电压跟随器U3B的同相输入端还连接有电容C50。
动植物组织消融仪还包括用于采集DCDC功率调节单元的可调电压的输出电压采样单元，采集的可调电压通过第二电压跟随器U3A发送至主控单元。
详细阐述输出电压采样单元的电路原理：串联在可调电压输出端的分压电阻R55和R60进行电压采样并通过RC滤波进行滤波后输入到第二电压跟随器U3A的同相输入端，第二电压跟随器U3A的正极电压端连接3.3V电压，第二电压跟随器U3A的输出端连接主控单元，其中，电阻R59和电容C39组成RC滤波，3.3V电压还连接有电容C44，第二电压跟随器U3A的输出端还连接有电容C43。
驱动单元包括第一驱动单元和第二驱动单元，其中，第一驱动单元包括第一驱动MOS管Q6，第二驱动单元包括第二驱动MOS管Q2。第一驱动单元的第二PWM信号接收端N接收主控单元发送的第一路第二PWM信号，第一驱动单元的输出端连接变压单元的原边线圈的同名端，第二驱动单元的第二PWM信号接收端P接收主控单元发送的第二路第二PWM信号，第二驱动单元的输出端连接变压单元的原边线圈的异名端，通过第二PWM信号接收端N接收的第一路第二PWM信号控制第一驱动MOS管Q6导通/截止，通过第二PWM信号接收端P接收的第二路第二PWM信号控制第二驱动MOS管Q2截止/导通。
详细阐述驱动单元和变压单元的电路原理：变压单元为推挽变压器，DCDC功率调节单元的可调电压输出端连接推挽变压器的原边第一线圈的异名端和第二线圈的同名端，电阻R10和R14串联于地与第二PWM信号接收端N之间，第一驱动MOS管Q6的栅极连接电阻R10和R14之间的节点，源极接地，漏极为第一驱动单元的输出端并连接推挽变压器的原边第一线圈的同名端；电阻R5和R13串联于地与第二PWM信号接收端P之间，第二驱动MOS管Q2的栅极连接电阻R5和R13之间的节点，源极接地，漏极为第二驱动单元的输出端并连接推挽变压器的原边第二线圈的异名端。
当第二PWM信号接收端N接收的第一路第二PWM信号为H且第二PWM信号接收端P接收的第二路第二PWM信号为L时，控制第一驱动MOS管Q6导通，第二驱动MOS管Q2截止，原边第一线圈的输入端回路接通并且输入电流方向为原边第一线圈的异名端至同名端，原边第二线圈的输入端回路截止，副边线圈的输出端回路的输出电流方向为副边线圈的同名端至异名端。
当第二PWM信号接收端N接收的第一路第二PWM信号为L且第二PWM信号接收端P接收的第二路第二PWM信号为H时，控制第一驱动MOS管Q6截止，第二驱动MOS管Q2导通，原边第二线圈的输入端回路接通并且输入电流方向为原边第二线圈的同名端至异名端，原边第一线圈的输入端回路截止，副边线圈的输出端回路的输出电流方向为副边线圈的异名端至同名端，将输入的直流电压转换成频率30KHz、电压上百伏特的交流波形，为后级谐振单元提供条件。
谐振单元设于推挽变压器的输出端回路中，谐振单元为LC串联谐振，包括串联于推挽变压器的输出端回路中的电感T1和电容C1。电感T1采用EFD20。
超声换能器设于推挽变压器的输出端回路中，本实施例中，通过接口J2连接超声换能器。
采样单元采集推挽变压器的输出端回路电压和电流，采样单元包括电压采样单元和电流采样单元。
电压采样单元采集推挽变压器的输出端回路电压，推挽变压器的输出端回路设有串联的采样电阻R1、R2、R3、R4和R15，电压采样端连接在电阻R4和R15之间节点，并将采集的电压信号发送至电压采样单元进行滤波、放大，然后发送至主控单元做谐振调节。
详细阐述电压采样单元的电路原理：采集的电压信号依次通过RC滤波进行滤波、电容C67进行隔直后输入到第一运放U7B的同相输入端，第一运放U7B的反相输入端与输出端之间连接负反馈电阻R63，第一运放U7B的输出端通过RC滤波进行滤波后，然后通过电阻R72输入到第一比较器U8A的同相输入端，串联于3.3V电压与地之间的分压电阻R77和R76进行电压采样输入到第一比较器U8A的反相输入端，第一比较器U8A的输出端通过电阻R71连接主控单元，电阻R72连接第一比较器U8A的同相输入端的另一端还通过电阻R70连接主控单元，其中，电阻R61和电容C65组成RC滤波对采集的电压信号进行滤波，电阻R62和电容C66组成RC滤波对第一运放U7B的输出端输出的信号进行滤波，第一运放U7B的反相输入端还串联有电阻R64和电容C70，第一运放U7B的同相输入端还连接有直流3.3V电压，该直流3.3V电压通过分压电阻R56、R58和R57分压后产生分压输入到第一运放U7B的同相输入端，分压还通过电容C64滤波，其中，电阻R58的一端连接第一运放U7B的同相输入端且另一端连接电阻R56，电阻R56连接3.3V电压，电阻R57的一端连接电阻R56和R58之间的节点且另一端接地，第一比较器U8A的正极电压端连接3.3V电压，该3.3V电压通过并联的电容C76和C77滤波。
电流采样单元采集推挽变压器的输出端回路电流，电流采样端串联在推挽变压器的输出端回路，并将采集的电流信号发送至电流采样单元进行滤波、放大，然后发送至主控单元做谐振调节。
详细阐述电流采样单元的电路原理：采集的电流信号依次通过RC滤波进行滤波、电容C75进行隔直后输入到第二运放U7A的同相输入端，第二运放U7A的反相输入端与输出端之间连接负反馈电阻R73，第二运放U7A的输出端通过RC滤波进行滤波后，然后通过电阻R81输入到第二比较器U8B的同相输入端，串联于3.3V电压与地之间的分压电阻R82和R83进行电压采样输入到第二比较器U8B的反相输入端，第二比较器U8B的输出端通过电阻R80连接主控单元，电阻R81连接第二比较器U8B的同相输入端的另一端还通过电阻R79连接主控单元，其中，电阻R65和电容C68组成RC滤波对采集的电流信号进行滤波，电阻R69和电容C69组成RC滤波对第二运放U7A的输出端输出的信号进行滤波，第二运放U7A的反相输入端还串联有电阻R74和电容C78，第二运放U7A的同相输入端还连接有直流3.3V电压，该直流3.3V电压通过分压电阻R78、R66、R67和R68分压后产生分压输入到第二运放U7A的同相输入端，分压通过电容C71滤波，其中，电阻R68的一端连接第二运放U7A的同相输入端且另一端连接串联于3.3V电压端的电阻R78和R66，电阻R67的一端连接电阻R66和R68之间的节点且另一端接地，第二比较器U8B的正极电压端通过电阻R78连接3.3V电压，该3.3V电压通过并联的电容C73和C74滤波。
主控单元，输出第一PWM信号控制DCDC功率调节单元输出可调电压，并通过采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元的第一驱动MOS管Q6和第二驱动MOS管Q2。
主控单元包括主控芯片U2和主控芯片外围电路，其中，主控芯片U2的型号为N32905U1DN，采用ARM9内核，主频200MHz，主控芯片外围电路包括系统时钟、复位等。
N32905U1DNN3290x基于ARM926EJ-S CPU内核，集成了JPEG编解码器，CMOS传感器接口，32通道SPU(声音处理单元)，ADC，DAC，可满足各种应用需求，同时节省BOM成本。ARM926@200MHz，同步DRAM，2D BitBLT加速器，CMOS图像传感器接口，LCD面板接口。N32905U1DNN3290x最大分辨率为XVGA(1,024x768)@TFT LCD面板。2D BitBLT加速器加速图形计算，使渲染平滑，卸载CPU，以节省功耗。
为了满足整体系统BOM成本的不同要求，不同尺寸的DRAM与N3290x主SoC堆叠成一个封装，即多芯片封装(MCP)。N32905U1DNN3290x特别采用1Mbitx16 3.3V SDRAM设计。N32905U1DNN3290x特别采用4Mbitx16 1.8V DDR SDRAM设计。一个16Mbitx16 1.8V DDR2SDRAM堆叠在N32905U1DNN3290x内部，以确保更高的性能并最大限度地减少系统设计工作，如EMI和噪声耦合。通过采用双层PCB以及消除阻尼电阻，EMI防护组件等，可以降低总BOM成本。
以上技术方案已经充分公开并可实施，本发明的动植物组织消融仪，利用第一PWM信号控制DCDC功率调节单元输出可调电压，使输出功率在0.5～1000W可调，利用采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元，使谐振超声频率为20-200KHz可调，调节谐振点到超声振子的共振点，使得超声振子工作在谐振状态，输出能量最大、振幅最强。
实施例2：
在实施例1的基础上，本实施例进一步公开了基于超声原理的动植物组织消融仪，在具体应用时，本发明的主控单元还包括辅助芯片U1和辅助芯片外围电路，辅助芯片U1的型号为STM32F031G4U6。
STM32F031G4U6的特征是：内核：
Figure PCTCN2020070559-appb-000001
32位
Figure PCTCN2020070559-appb-000002
CPU，频率高达48MHz；存储：16至32KB的闪存，带有硬件奇偶校验的4KB SRAM；CRC计算单元；重置和电源管理数字和I/O电源：2.0至3.6V，模拟电源：VDDA＝从VDD到3.6V，上电/掉电复位(POR/PDR)，可编程电压检测器(PVD)，低功耗模式：睡眠，停止和支持，用于RTC和备份寄存器的VBAT电源；时钟管理：4至32MHz晶体振荡器，用于带校准的RTC的32kHz振荡器，具有x6PLL选项的内部8MHz RC，内部40kHz RC振荡器；最多39个快速I/O，所有可映射到外部中断向量，最多26个I/O，具有5V容限；5通道DMA控制器；1×12位，1.0μsADC(最多10个通道)，转换范围：0至3.6V，将模拟电源从2.4分离到3.6V；最多9个计时器，1x16位7通道高级控制定时器，用于6通道PWM输出，具有死区时间，发电和紧急停止，1x32位和1x16位定时器，最多4个IC/OC，可用于IR控制解码，1x16位定时器，带2个IC/OC，1个OCN，死区时间和紧急停止，1x16位定时器，带IC/OC和OCN，死区时间，紧急停止和用于IR控制的调制器门，具有1个IC/OC的1x16位定时器，独立和系统看门狗定时器，SysTick定时器：24位向下计数器；日历RTC，具有警报和定期唤醒功能从停止/待机；通信接口1个I2C接口，支持快速模式加(1Mbit/s)，20mA电流吸收，SMBus/PMBus，从Stop唤醒模式，1x USART支持主同步，SPI和调制解调器控制，ISO7816接口，LIN，IrDA功 能，自动波特率速率检测和唤醒功能，1x SPI(18Mbit/s)，4至16可编程位帧，带I2S接口；串行线调试(SWD)；96位唯一ID；扩展温度范围：-40至+105℃；所有包装
Figure PCTCN2020070559-appb-000003
2。
主控单元的主控芯片U2通过采集变压单元的输出端回路电压和电流的采样单元的反馈，向辅助芯片U1发送指令，辅助芯片U1接收主控芯片U2的指令，输出两路频率为30KHz，占空比互补的第二PWM信号至驱动单元的第一驱动MOS管Q6和第二驱动MOS管Q2，同时，两路频率为30KHz，占空比互补的第二PWM信号还反馈至主控芯片U2。同时，第一比较器U8A的输出端和第二比较器U8B的输出端输出的信号发送至辅助芯片U1。
两路频率为30KHz，占空比互补的第二PWM信号还分别通过第一信号放大输出电路和第二信号放大输出电路进行放大后发送至驱动单元的第一驱动MOS管Q6和第二驱动MOS管Q2。
详细阐述第一信号放大输出电路的电路原理：第二PWM信号通过串联的分压电阻R49和R53输入第一放大三极管Q5的基极进行放大，第一放大三极管Q5的集电极通过电阻R34连接12V电压，第一放大三极管Q5的发射极接地，第一放大三极管Q5的集电极与第一输出三极管Q2和第二输出三极管Q4的基极连接，第一输出三极管Q2为P型三极管，第二输出三极管Q4为N型三极管，第一输出三极管Q2的集电极连接12V电压，放大后的第一路第二PWM信号从第一输出三极管Q2的发射极与第二输出三极管Q4的发射极连接节点处输出至第一驱动MOS管Q6的第二PWM信号接收端N。
当第二PWM信号输入为H时，第一输出三极管Q2导通，第二输出三极管Q4截止；当第二PWM信号输入为L时，第一输出三极管Q2截止，第二输出三极管Q4导通，由此输出放大后的方波第一路第二PWM信号。
详细阐述第二信号放大输出电路的电路原理：第二PWM信号通过串联的分压电阻R86和R87输入第二放大三极管Q8的基极进行放大，第二放大三极管Q8的集电极通过电阻R84连接12V电压，第二放大三极管Q8的发射极接地，第二放大三极管Q8的集电极与第三输出三极管Q6和第四输出三极管Q7的基极连接，第三输出三极管Q6为P型三极管，第四输出三极管Q7为N型三极管，第三输出三极管Q6的集电极连接12V电压，放大后的第二路第二PWM信号从第三输出三极管Q6的发射极与第四输出三极管Q7的发射极连接节点处输出至第二驱动MOS管Q2的第二PWM信号接收端P。
当第二PWM信号输入为H时，第三输出三极管Q6导通，第四输出三极管Q7截止；当第二PWM信号输入为L时，第三输出三极管Q6截止，第四输出三极管Q7导通，由此输出放大后的方波第二路第二PWM信号。
主控芯片U2还连接有显示单元，显示单元包括显示芯片J8和与显示芯片J8连接的LCD显示屏，该显示芯片J8的型号为FPC050，显示芯片J8连接3.3V电压，主控芯片U2的信号LCD_BL通过MOS管Q1放大后发送至显示芯片J8。
主控芯片U2还连接有按键，本实施例包括四个按键，分别为第一按键、第二按键、第三按键和第四按键，第一按键为左按键，第二按键为右按键，第三按键为中按键，第四按键为OK按键。其中，第一按键包括上拉电阻R4和下拉电阻R11，上拉电阻R4连接3.3V电压，上拉电阻R4和下拉电阻R11之间的节点连接按键S2的一端，按键S2的另一端接地，下拉电阻R11的另一端连接主控芯片U2，当按下按键S2，下拉电阻R11只有1K，第一按键输出低电平，当松开按键S2，输出被上拉电阻R4和下拉电阻R11上拉，第一按键输出高电平；第二按键包 括上拉电阻R6和下拉电阻R19，上拉电阻R6连接3.3V电压，上拉电阻R6和下拉电阻R19之间的节点连接按键S3的一端，按键S3的另一端接地，下拉电阻R19的另一端连接主控芯片U2，当按下按键S3，第二按键输出低电平，当松开按键S3，输出被上拉电阻R6和下拉电阻R19上拉，第二按键输出高电平；第三按键包括上拉电阻R8和下拉电阻R20，上拉电阻R8连接3.3V电压，上拉电阻R8和下拉电阻R20之间的节点连接按键S4的一端，按键S4的另一端接地，下拉电阻R20的另一端连接主控芯片U2，当按下按键S4，第三按键输出低电平，当松开按键S4，输出被上拉电阻R8和下拉电阻R20上拉，第三按键输出高电平；第四按键包括上拉电阻R10和下拉电阻R26，上拉电阻R10连接3.3V电压，上拉电阻R10和下拉电阻R26之间的节点连接按键S5的一端，按键S5的另一端接地，下拉电阻R26的另一端连接主控芯片U2，当按下按键S5，第四按键输出低电平，当松开按键S5，输出被上拉电阻R10和下拉电阻R26上拉，第四按键输出高电平。
主控芯片U2还连接有存储器，存储器采用SPI-FLASH器件，用于存储参数，包括谐振参数、设置参数等。
主控芯片U2还连接有USB接口单元，USB接口单元包括与主控芯片U2连接的USB芯片ESD1和与USB芯片ESD1连接的接口J7，USB芯片ESD1的型号为USBLC6，接口J7的型号为SIP254。
主控芯片U2还连接有触摸单元，触摸单元包括串联在3.3V电压与地之间的分压电阻R99和R101，连接电阻R99和R101之间节点的电阻R100以及基极连接电阻R99和R101之间节点的三极管Q10，三极管Q10的集电极与3.3V电压之间串联有分压电阻R98和R96，分压电阻R98和R96之间的节点连接MOS管Q9的栅极，MOS管Q9的源极连接3.3V电压，MOS管Q9的漏极连接触摸接口J1，触摸接口J1与主控芯片U2连接，触摸接口J1连接有触摸板，主控芯片U2发送的电源使能信号TP_PWEN通过电阻R100至三极管Q10的基极，当电源使能信号TP_PWEN为H时，三极管Q10导通，MOS管Q9导通。
主控芯片U2还连接有外部接口J4。
主控芯片U2还连接有蜂鸣器。
实施例3：
本实施例提供一种提取液，包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍，并通过乙酸调节提取液的pH到4。将该提取液应用到用于保存组织或细胞。
实施例4：
本实施例提供一种提取液，包括：
Figure PCTCN2020070559-appb-000004
其中，本提取液通过乙酸调节pH至4。将该提取液应用在提取RNA中。
实施例5：
本实施例提供一种提取液，包括提取液A和提取液B；
其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油和0.01mol/L的SDS；余量为溶剂。
所述提取液A和提取液B的pH均为4。
采用本实施例中的提取液提取到的RNA量在1000-1800μg/mL，测得A260/A280在1.6-1.9范围，A260/A230在1.6-1.9范围。琼脂糖凝胶电泳图片如图7所示。
采用上述提取液A和提取液B以及利用实施例1或实施例2基于超声原理的动植物组织消融仪快速提取RNA的方法，包括步骤：1.先用提取液A保存组织或细胞，所述提取液A包括1.2-2.0mol/L的硫氰酸胍和0.5-1.5mol/L的盐酸胍，且pH为4；
2.再向提取液A保存的组织或细胞中加入提取液B，颠倒混匀，获得混合物，所述提取液B包括以体积分数计的30-65vt％的水饱和苯酚和9-15vt％的甘油，余量为溶剂；
提取液B还包括以摩尔浓度计的0.20-1mol/L的硫氰酸胍、0.4-0.8mol/L的硫氰酸铵和0.0073-0.018mol/L的表面活性剂，且pH调节为4；
3.从组织中提取RNA时，将步骤2得到的混合物利用基于超声原理的动植物组织消融仪进行超声破碎；从细胞中提取RNA时，直接进行下述步骤5中的操作；
4.将超声破碎后的混合物颠倒混匀，其中超声功率为0.5-1000W，超声时间为0.5-30s；
5.在步骤3从细胞提取RNA的混合物或步骤4的混合物中，加入氯仿，混匀后离心，取上层液体；
6.在上层液体中加入异丙醇，混匀后离心，弃去液体，获得沉淀物；
7.向沉淀物中加入乙醇溶液，混匀后离心，弃去液体；
8.待乙醇挥发后加入水溶解。
实验1：
取8个1.5mlEP管，每个EP管加入4mg大鼠肝脏组织，对每个EP管进行编号，其中，1、3、5、7号EP管分别加入250ul提取液A，于室温下保存48h，2、4、6、8号EP管分别加入250ulRNAlater溶液，于室温下保存48h。
再向上述所有EP管中分别加入750ul提取液B。
将上述1-8号EP管颠倒混匀后进行超声消融，超声时间为5s，频率为20kHz，功率为100W；将超声后的EP管颠倒几次，使其中的混合物混匀；向处理后的EP管中分别加入200ul氯仿，剧烈颠倒混匀30s后，在10000g的离心力下离心1min，取上层液体；将获得的上层液体分别倒入另一批对应编号的干净EP管中，然后分别加入500ul异丙醇，缓慢颠倒混匀50s后，在10000g的离心力下离心1min，弃去液体，获得沉淀物；向上述沉淀物中分别加入600ul75vt％乙醇溶液，缓慢颠倒混匀30s后，在10000g的离心力下离心2min，弃去液体；将的EP管置于空气中干燥5min后加入40ulddH2O溶解；将获得的RNA进行微量紫外分光光度计测定OD值以及琼脂糖凝胶电泳检测。
得到实验1数据并进行结果分析，其得到结果可证明采用提取液A相较于现有的RNAlater溶液能够得到质量更好的RNA提取纯度。
本次使用本发明的A液，增加和延长了组织保存时间，采用本发明的A液，与现在市面上RNA提取试剂Trizol，对保存效果进行对比，分别使用以上两种试剂对大鼠脾脏组织进行保存，存放于室温和37℃两种，保存时间设置在7天以上，后对保存后组织形态进行观察，发现Trizol保存的脾脏组织已全部溶解，我方发明的A液保存组织正常，后对两种保存的脾脏组织进行RNA提取，结果本实施例的A液提取到的RNA量足于高出Trizol保存组织的3倍水平以上。
本发明与Trizol保存的脾脏组织后提取结果进行对比，结果trizol保存后的组织提取到的RNA量在474μg/mL，测得A260/A280在1.583上下，A260/A230在2.563上下。使用本发明的RNAlater提取到的RNA量在1241μg/mL，测得A260/A280在1.8上下，A260/A230在2.2上下。
实验2：
同样取8个1.5mlEP管，每个EP管加入4mg大鼠肝脏组织，对每个EP管进行编号，其中，所有EP管分别加入250ulRNAlater溶液，于室温下保存48h。
再向上述其中的1、3、5、7号EP管中分别加入750ul提取液B；而向2、4、6、8号EP管分别加入1ml的TRIzol溶液。
将上述1-8号EP管颠倒混匀后进行超声消融，超声时间为5s，频率为20kHz，功率为100W；将超声后的EP管颠倒几次，使其中的混合物混匀；向处理后的EP管中分别加入200ul氯仿，剧烈颠倒混匀30s后，在10000g的离心力下离心1min，取上层液体；将获得的上层液体分别倒入另一批对应编号的干净EP管中，然后分别加入500ul异丙醇，缓慢颠倒混匀50s后，在10000g的离心力下离心1min，弃去液体，获得沉淀物；向上述沉淀物中分别加入600ul75vt％乙醇溶液，缓慢颠倒混匀30s后，在10000g的离心力下离心2min，弃去液体；将的EP管置于空气中干燥5min后加入40ulddH2O溶解；将获得的RNA进行微量紫外分光光度计测定OD值以及琼脂糖凝胶电泳检测。
得到实验1数据并进行结果分析，其得到结果可证明采用提取液B相较于现有的TRIzol溶液能够得到质量更好的RNA提取纯度。
本实施例提取液A与提取液B混合后提取到的RNA相较于现有Trizol溶液提取到更好质量的RNA，采用以上两种试剂对大鼠脾脏组织进行RNA提取，对提取后的RNA进行浓度测定，Trizol提取到的RNA量一般处于400μg/mL，同时对A260/A230进行测定，测定值一般在 1.5水平。A260/A280测定结果在1.8水平。而本实施例的试剂提取到的RNA量在1200μg/mL以上水平，同时对A260/A230进行测定，测定值一般在1.8水平。A260/A280测定结果在1.7水平。
实验3：
取8个1.5mlEP管，每个EP管加入4mg大鼠肝脏组织，对每个EP管进行编号，其中，1、3、5、7号EP管分别加入250ul提取液A，于室温下保存48h，2、4、6、8号EP管分别加入250ulRNAlater溶液，于室温下保存48h。
向上述1、3、5、7号EP管中分别加入750ul提取液B；弃去上述2、4、6、8号EP管中的RNA later溶液，加入1×PBS洗涤3次后弃液，再分别加入1ml的TRIzol溶液。
将上述1-8号EP管颠倒混匀后进行超声消融，超声时间为5s，频率为20kHz，功率为100W；将超声后的EP管颠倒几次，使其中的混合物混匀；向处理后的EP管中分别加入200ul氯仿，剧烈颠倒混匀30s后，在10000g的离心力下离心1min，取上层液体；将获得的上层液体分别倒入另一批对应编号的干净EP管中，然后分别加入500ul异丙醇，缓慢颠倒混匀50s后，在10000g的离心力下离心1min，弃去液体，获得沉淀物；向上述沉淀物中分别加入600ul75vt％乙醇溶液，缓慢颠倒混匀30s后，在10000g的离心力下离心2min，弃去液体；将的EP管置于空气中干燥5min后加入40ulddH2O溶解；将获得的RNA进行微量紫外分光光度计测定OD值以及琼脂糖凝胶电泳检测。
得到实验1数据并进行结果分析，其得到结果可证明采用提取液A相较于现有的RNAlater溶液能够得到质量更好的RNA提取纯度。
采用本实施例的保存A液加裂解液B混合模式，提取到的RNA提取纯度高，采用1％琼脂糖凝胶电泳对我方试剂提取到的RNA进行确证，本实施例试剂提取到的RNA质量及效果均好于trizol提取到的RNA。二者比较结果为Trizol提取到的RNA量一般处于400μg/mL，同时对A260/A230进行测定，测定值一般在1.5水平。A260/A280测定结果在1.8水平。而本实施例的试剂提取到的RNA量在1200μg/mL以上水平，同时对A260/A230进行测定，测定值一般在1.8水平。A260/A280测定结果在1.7水平。
实验4：
取6个1.5mlEP管，每个EP管加入约106个Panc-1细胞，对每个EP管进行编号，其中，1、2、3号EP管分别加入250ul提取液A和750ul提取液B，4、5、6号EP管分别加入1mlTRIzol；将上述EP管颠倒几次，使其中的混合物混匀；在上述EP管中分别加入200ul氯仿，剧烈颠倒混匀10s后，在10000g的离心力下离心1min，取上层液体；
将获得的上层液体分别倒入另一批对应编号的干净EP管中，然后分别加入500ul异丙醇，缓慢颠倒混匀50s后，在10000g的离心力下离心1min，弃去液体，获得沉淀物；向上述沉淀物中分别加入600ul75vt％乙醇溶液，缓慢颠倒混匀30s后，在10000g的离心力下离心1min，弃去液体；将EP管置于空气中干燥5min后加入40ulddH2O溶解；将获得的RNA进行微量紫外分光光度计测定OD值以及琼脂糖凝胶电泳检测。
采用本实施例的试剂配方，进行RNA提取以及质量和纯度评价，从分光光度计测定的结果及琼脂糖凝胶电泳结果进行比对，本实施例结果提取到的RNA量在1200μg/mL以上水平，同时对A260/A230进行测定，测定值一般在1.8水平。A260/A280测定结果在1.7水平。
实施例6：
本实施例提供一种提取液，包括提取液A和提取液B；
其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均用乙酸调至4。
并且针对上述最佳方法，通过设置多组对比组以控制变量方式进行实验。
实验组1：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均用乙酸调至4。
实验组2：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均用乙酸调至4。
实验组3：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均为7或2。
实验组4：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均通过乙酸调至4。
实验组5：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的 硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均通过乙酸调至4。
实验组6：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均通过乙酸调至4。
实验组7：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均通过乙酸调至4。
实验组8：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、0.15mol/L的乙酸钠、8vt％的异丙醇和0.1wt％的8-羟基喹啉。
提取液A和提取液B的pH均通过乙酸调至4。
实验组9：
本实施例提供一种提取液，包括提取液A和提取液B；其中提取液A包括1.6mol/L的硫氰酸胍和1mol/L的盐酸胍；
而提取液B包括57vt％的水饱和苯酚、1mmol/L的DTT、0.24mol/L的硫氰酸胍、0.6mol/L的硫氰酸铵、11.25vt％的甘油、0.3wt％的SDS、0.1wt％的β-巯基乙醇、0.08mmol/L的柠檬酸钾、0.1wt‰的溴酚蓝、5mol/L的尿素、0.15mol/L的乙酸钠、8vt％的异丙醇、0.1wt％的8-羟基喹啉和0.1mmol/L的EDTA。
所述提取液A和提取液B的pH均通过乙酸调至4。
针对上述缺少和增加不同组分的实验组进行统一的实验验证，每组均取8个1.5mlEP管，每个EP管加入4mg大鼠肝脏组织，对每个EP管进行编号，其中，所有EP管分别加入250ul提取液A，于室温下分别保存48h。
并向上述所有EP管中分别加入750ul提取液B。
将上述1-8号EP管颠倒混匀后进行超声消融，超声时间为5s，频率为20kHz，功率为 100W；将超声后的EP管颠倒几次，使其中的混合物混匀；向处理后的EP管中分别加入200ul氯仿，剧烈颠倒混匀30s后，在10000g的离心力下离心1min，取上层液体；将获得的上层液体分别倒入另一批对应编号的干净EP管中，然后分别加入500ul异丙醇，缓慢颠倒混匀50s后，在10000g的离心力下离心1min，弃去液体，获得沉淀物；向上述沉淀物中分别加入600ul75vt％乙醇溶液，缓慢颠倒混匀30s后，在10000g的离心力下离心2min，弃去液体；将的EP管置于空气中干燥5min后加入40ulddH2O溶解；将获得的RNA进行微量紫外分光光度计测定OD值以及琼脂糖凝胶电泳检测。
结果分析如下：实验组1因为缺少硫氰酸胍直接导致裂解不充分，影响到RNA提取的常量。提取到的RNA量明显减少，与原配方提取结果对比，缺少0.24mol/L的硫氰酸胍试剂提取到的RNA量偏少，提取到的RNA量将在300μg/mL上下，A260/A230在1.7上下，A260/A280在2以上，采用分光光度计进行定量测定，采用琼脂糖凝胶电泳对提取到的RNA纯度进行判定。实验组2因为缺少0.3wt％的SDS直接导致裂解不充分，影响到RNA提取的常量。提取到的RNA量明显减少，与原配方提取结果对比，缺少0.3wt％的SDS试剂提取到的RNA量偏少，SDS直接影响裂解，采用分光光度计进行定量测定，采用琼脂糖凝胶电泳对提取到的RNA纯度进行判定，RNA的产量在200μg/mL上下。
实验组3因为pH值改变，既影响RNA产量，同时导致提取到的RNA不纯，引入了基因组污染。采用琼脂糖凝胶电泳对提取到的RNA纯度进行比较，可以发现改变pH值的试剂，均有基因组污染。pH值改变，直接会导致基因组污染，不会影响RNA的产量。实验组4因为β-巯基乙醇做为还原剂，确保欢迎液中的硫酸铵不被氧化，保证试剂稳定性，缺少0.1wt％的β-巯基乙醇试剂将直接导致试剂保存不稳定，存放时间久后会导致试剂颜色发生改变。实验组5因为缺少0.08mmol/L的柠檬酸钾，将会影响到试剂缓冲体系，从而影响实验结果。而实验组6缺少8-羟基喹啉，因为8-羟基喹啉作用是保持水饱和酚的稳定性，防止水饱和酚的氧化。影响到B液长时间保存及稳定性。实验组7中因为增加糖污染，影响RNA提取质量。而实验组8中所述缺少EDTA会增加提取到RNA被RNase降解的风险。因为5mol/L的尿素和1mmol/L的DTT均是抑制RNase的作用，使得提取出来的RNA免遭降解。故实验组9中保存的RNA已经降解。
本发明不局限于上述可选实施方式，任何人在本发明的启示下都可得出其他各种形式的产品，但不论在其形状或结构上作任何变化，凡是落入本发明权利要求界定范围内的技术方案，均落在本发明的保护范围之内。

Claims (28)

  1. 利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：包括步骤：
    G1.先用提取液A保存组织或细胞，所述提取液A包括1.2-2.0mol/L的硫氰酸胍和0.5-1.5mol/L的盐酸胍，且pH为4；
    G2.再向提取液A保存的组织或细胞中加入提取液B，颠倒混匀，获得混合物，所述提取液B包括以体积分数计的30-65vt％的水饱和苯酚和9-15vt％的甘油，余量为溶剂；
    提取液B还包括以摩尔浓度计的0.20-1mol/L的硫氰酸胍、0.4-0.8mol/L的硫氰酸铵和0.0073-0.018mol/L的表面活性剂，且pH调节为4；
    G3.从组织中提取RNA时，将步骤G2得到的混合物利用基于超声原理的动植物组织消融仪进行超声破碎；从细胞中提取RNA时，直接进行下述步骤G5中的操作；
    G4.将超声破碎后的混合物颠倒混匀；
    G5.在步骤G3从细胞提取RNA的混合物或步骤G4的混合物中，加入氯仿，混匀后离心，取上层液体；
    G6.在上层液体中加入异丙醇，混匀后离心，弃去液体，获得沉淀物；
    G7.向沉淀物中加入乙醇溶液，混匀后离心，弃去液体；
    G8.待乙醇挥发后加入水溶解；
    所述基于超声原理的动植物组织消融仪，包括：电源、DCDC转换单元、主控单元、DCDC功率调节单元、驱动单元、变压单元、谐振单元、超声换能器和采样单元；
    所述电源，通过DCDC转换单元和DCDC功率调节单元转换电压后给动植物组织消融仪使用；主控单元，输出第一PWM信号控制DCDC功率调节单元输出可调电压，并通过采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元并经过变压单元变压，然后通过设于变压单元的输出端回路中的谐振单元调节谐振点到超声振子的共振点，使得设于变压单元的输出端回路中的超声振子工作在谐振状态。
  2. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中的表面活性剂为十二烷基硫酸钠。
  3. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液A采用盐酸调节pH值，所述提取液B采用乙酸调节pH值。
  4. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为6.4mmol/L-19.20mmol/L的β-巯基乙醇。
  5. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为0.05-0.15mmol/L的柠檬酸钾。
  6. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为74.62μmol/L-223.87μmol/L的溴酚蓝。
  7. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于，所述提取液B中还包括摩尔浓度为0.01-2mol/L的乙酸钠。
  8. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括体积分数为5-25％的异丙醇。
  9. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为0.689mmol/L-68.9mmol/L的8-羟基喹啉。
  10. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为0.5-1000mmol/L的DTT。
  11. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为0.5-5mol/L的尿素。
  12. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为0.1-500mmol/L的EDTA。
  13. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液B中还包括摩尔浓度为0.01-10mmol/L的乙酸铵。
  14. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述步骤G8中的水为双蒸水、去离子水或超纯水。
  15. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述提取液A和提取液B的总质量为组织或细胞质量的100-150倍。
  16. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述步骤G3中，超声功率为0.5-1000W，超声时间为0.5-30s。
  17. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述步骤G5中，氯仿的体积为混合物体积的0.1-0.4倍。
  18. 根据权利要求1-3任意一项所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述步骤G6中，异丙醇与上层液体的体积比为1：1。
  19. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的DCDC功率调节单元包括：开关控制信号接收端连接主控单元接收开关控制信号的开关控制信号接收电路；
    第一PWM信号接收端连接主控单元接收第一PWM信号的第一PWM信号接收电路；
    通过主控单元发送的开关控制信号和第一PWM信号将电源转换为可调电压输出的第四DCDC转换单元。
  20. 根据权利要求19所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的第四DCDC转换单元包括：依次处理电源电压的第四电压输入滤波、第四电压转换芯片U1和第四电压输出滤波，第四电压输入滤波包括连接在电源端的滤波电容，第四电压转换芯片U1的上管驱动信号参考点管脚SW通过储能电感L1连接到可调电压输出端，串联在可调电压输出端的分压电阻R32和R47进行电压采样输入到第四电压转换芯片U1的参考电压管脚FB，第四电压转换芯片U1的上管驱动信号参考点管脚SW还连接有续流二极管D2，使能管脚EN还连接有电阻R33，电阻器时序/外部时钟管脚RT/CLK还连接有频率分压电阻R85，频率补偿管脚COMP还连接有用于调节环路、稳定电压输出的电容C51、C49和电阻R46，其中，电容C49与串联的电容C51和电阻R46并联，第四电压输出滤波包括连接在可调电压输出端的滤波电容；
    开关控制信号接收电路包括：串联于转换后电压与开关控制信号接收端之间的电阻 R104和R105，发射极连接转换后电压、基极连接电阻R104和R105之间的节点且集电极连接第四电压转换芯片U1的使能管脚EN的三极管Q12，当开关控制信号为L时，三极管Q12导通，第四电压转换芯片U1开启工作；
    第一PWM信号接收电路包括：依次处理第一PWM信号的RC滤波和第一电压跟随器U3B，第一PWM信号接收端接收的第一PWM信号通过RC滤波进行滤波后输入到第一电压跟随器U3B的同相输入端，第一电压跟随器U3B的输出端通过分压电阻R54连接到分压电阻R32和R47之间的节点。
  21. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的驱动单元包括第一驱动单元和第二驱动单元，第一驱动单元的第二PWM信号接收端N接收主控单元发送的第一路第二PWM信号，第一驱动单元的输出端连接变压单元的原边线圈的同名端，第二驱动单元的第二PWM信号接收端P接收主控单元发送的第二路第二PWM信号，第二驱动单元的输出端连接变压单元的原边线圈的异名端，通过第二PWM信号接收端N接收的第一路第二PWM信号控制第一驱动MOS管Q6导通/截止，通过第二PWM信号接收端P接收的第二路第二PWM信号控制第二驱动MOS管Q2截止/导通。
  22. 根据权利要求21所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的变压单元为推挽变压器，DCDC功率调节单元的可调电压输出端连接推挽变压器的原边第一线圈的异名端和第二线圈的同名端；
    所述第一驱动单元包括串联于地与第二PWM信号接收端N之间的电阻R10和R14，栅极连接电阻R10和R14之间的节点、源极接地且漏极为第一驱动单元的输出端并连接推挽变压器的原边第一线圈的同名端的第一驱动MOS管Q6；
    所述第二驱动单元包括串联于地与第二PWM信号接收端P之间的电阻R5和R13，栅极连接电阻R5和R13之间的节点、源极接地且漏极为第二驱动单元的输出端并连接推挽变压器的原边第二线圈的异名端的第二驱动MOS管Q2。
  23. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的谐振单元为LC串联谐振，LC串联谐振包括串联于变压单元的输出端回路中的电感T1和电容C1。
  24. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的采样单元包括采集变压单元的输出端回路电压的电压采样单元和采集变压单元的输出端回路电流的电流采样单元，相应地，变压单元的输出端回路设有多个串联的采样电阻；通过连接在多个采样电阻之间节点的电压采样端采集电压信号然后发送至电压采样单元进行滤波和放大，然后发送至主控单元做谐振调节；通过串联在变压单元的输出端回路的电流采样端采集电流信号发送至电流采样单元进行滤波和放大，然后发送至主控单元做谐振调节。
  25. 根据权利要求1所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪的主控单元包括主控芯片U2和主控芯片外围电路，主控芯片U2输出第一PWM信号控制DCDC功率调节单元输出可调电压，并通过采集变压单元的输出端回路电压和电流的采样单元的反馈，输出两路占空比互补的第二PWM信号至驱动单元。
  26. 根据权利要求25所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述主控单元还包括辅助芯片U1和辅助芯片外围电路，主控单元的主控芯片U2通过采集变压单元的输出端回路电压和电流的采样单元的反馈，向辅助芯片U1发送指令，辅助芯片U1接收主控芯片U2的指令，输出两路占空比互补的第二PWM信号至驱动单元，同时，两路占空比互补的第二PWM信号还反馈至主控芯片U2。
  27. 根据权利要求25所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述两路占空比互补的第二PWM信号还分别通过第一信号放大输出电路和第二信号放大输出电路进行放大后发送至驱动单元。
  28. 根据权利要求25所述的利用基于超声原理的动植物组织消融仪快速提取RNA的方法，其特征在于：所述动植物组织消融仪还包括与主控芯片U2连接的显示单元、按键、存储器、USB接口单元或/和触摸单元。
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