WO2021226917A1 - Novel method of one-step whole transcriptome amplification - Google Patents
Novel method of one-step whole transcriptome amplification Download PDFInfo
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- WO2021226917A1 WO2021226917A1 PCT/CN2020/090186 CN2020090186W WO2021226917A1 WO 2021226917 A1 WO2021226917 A1 WO 2021226917A1 CN 2020090186 W CN2020090186 W CN 2020090186W WO 2021226917 A1 WO2021226917 A1 WO 2021226917A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- the invention involves methods and reagents for a one-step whole transcriptome amplification method that combines different reaction steps in the same reaction system.
- the method can be used to analyze RNA from single cells or minimal amount of start materials.
- Cell is the smallest functional unit of biological organism. Cellular heterogeneity plays an important role in the development as well as diseases. There have been increasing needs to analyze genome or transcriptome at single cell level to gain more accurate insights into the complex biological systems.
- a typical human cell has less than 10pg DNA and 10-20pg total RNA.
- Current sequencing methods require at least nanogram amount of nucleic acid to start with. Therefore, effective nucleic acid amplification methods are required to enable single cell sequencing.
- SMART Switching Mechanism At 5’ end of the RNA Transcript
- SMART Switching Mechanism At 5’ end of the RNA Transcript
- the one-step method of the present invention performs low-start-up reverse transcription, the operation is simple and easy, which effectively streamlines the process, reduces risk of sample loss and cross contamination, and makes it easy to automate the whole process.
- This invention in the traditional method, if you want to get the transcriptome information of a cells, you need to go through 4 steps, 1 cell lysis, 2 reverse transcription, 3 cDNA amplification , 4 library sequencing,
- This invention incluede a novel whole transcriptome amplification method that can turn few cells or minimal amount of RNA to whole length cDNA library in a convenient one-tube reaction by using the same optimized reaction mix to facilitate RT, template switching, and PCR amplification. so that the first three steps of the traditional method can be completed in one step.
- This invention include a novel whole transcriptome amplification method that can turn few cells or minimal amount of RNA to fill length cDNA library in a convenient one-tube reaction by using the same optimized reaction mix to facilitate RT, template switching, and PCR amplification.
- our method after lysing the cells by temperature in tube we directly conduct reverse transcription experiments. After reverse transcription, we adjust the temperature to 95 °C to activate the hot-start enzyme and then perform PCR amplification reactions. so that the first three steps of the traditional method can be completed in one step., the operation is simple and easy, which effectively streamlines the process, reduces risk of sample loss and cross contamination, and makes it easy to automate the whole process.
- Fig1 Fragment distribution of L1.
- Fig 5 Distribution of library construction fragments of L1.
- Fig 7 Distribution of library construction fragments of L3.
- Fig 8 Distribution of library construction fragments of L3.
- Fig 9 Gene expression of Ll, L2, L3, and L4.
- One embodiment of the invention is to combine enzymes, dNTPs, oligos, and buffers required for cell lysis, reverse transcription, template switching, PCR amplification in one reaction mix. Once a single cell, or limited number of cells, is added to the reaction, cell can be first lyzed, the RNA released and reverse transcribed to cDNA, and a sequence tag is added to the 3’ of cDNA by template switching. The cDNA is then amplified in PCR. Different enzymatic reactions can be performed in the same master mix at different temperatures.
- Step 1 Reaction Vol ⁇ Me Run Time 42 °C 20 ⁇ L 1: 30: 00
- step 2 Reaction Vol ⁇ Me Run Time Lid Temperature 82°C 72°C 20 ⁇ L 0: 10: 00
- PCR amplification after the reverse transcription reaction Divide the above 20 ⁇ l product into 2 centrifuge tubes, each 10 ⁇ l, prepare the following system.
- VAHTS DNA Clean Beads (Vazyme N411-01 /02/03)
- the present invention has been verified by Confirmatory experiment which have proved the feasibility of the method. Now we list the results of a comparative experiment we have done. Although the one-step method is less efficient than the conventional method, it does not affect the later library construction experiment.
Abstract
Provided are methods and reagents for a one-step whole transcriptome amplification method that combines different reaction steps in the same reaction system.
Description
The invention involves methods and reagents for a one-step whole transcriptome amplification method that combines different reaction steps in the same reaction system. The method can be used to analyze RNA from single cells or minimal amount of start materials.
Cell is the smallest functional unit of biological organism. Cellular heterogeneity plays an important role in the development as well as diseases. There have been increasing needs to analyze genome or transcriptome at single cell level to gain more accurate insights into the complex biological systems. A typical human cell has less than 10pg DNA and 10-20pg total RNA. Current sequencing methods require at least nanogram amount of nucleic acid to start with. Therefore, effective nucleic acid amplification methods are required to enable single cell sequencing.
Conventional transcriptome amplification methods have multiple reaction steps, such as cell lysis, reverse transcription to synthesize cDNA, and cDNA amplification. For example, SMART (Switching Mechanism At 5’ end of the RNA Transcript) is the most widely used method for constructing Whole-length cDNAs from single cell or small amounts of RNA which relies on template switching to add a sequence tag to the 3’of cDNA following reverse transcription, followed by PCR reaction to amplify cDNA.
Current SMART procedure normally has three reaction steps: cell lysis; reverse transcription; and PCR amplification, that are performed in seprate reaction systems. The reaction mix needs to be transferred from one reaction container to the other one after each strp, introducing risk of sample loss and cross contamination.
Here we describe a novel whole transcriptome amplification method that can turn few cells or minimal amount of RNA to whole length cDNA library in a convenient one-tube reaction by using the same optimized reaction mix to facilitate RT, template switching, and PCR amplification. The one-step method of the present invention performs low-start-up reverse transcription, the operation is simple and easy, which effectively streamlines the process, reduces risk of sample loss and cross contamination, and makes it easy to automate the whole process.
Summary
In the traditional method, if you want to get the transcriptome information of a cells, you need to go through 4 steps, 1 cell lysis, 2 reverse transcription, 3 cDNA amplification , 4 library sequencing, This invention incluede a novel whole transcriptome amplification method that can turn few cells or minimal amount of RNA to whole length cDNA library in a convenient one-tube reaction by using the same optimized reaction mix to facilitate RT, template switching, and PCR amplification. so that the first three steps of the traditional method can be completed in one step.
In the traditional method, if you want to get the transcriptome information of a cells, you need to go through 4 steps, 1 cell lysis, 2 reverse transcription, 3 cDNA amplification, 4 library sequencing. This invention include a novel whole transcriptome amplification method that can turn few cells or minimal amount of RNA to fill length cDNA library in a convenient one-tube reaction by using the same optimized reaction mix to facilitate RT, template switching, and PCR amplification. In our method, after lysing the cells by temperature in tube we directly conduct reverse transcription experiments. After reverse transcription, we adjust the temperature to 95 ℃ to activate the hot-start enzyme and then perform PCR amplification reactions. so that the first three steps of the traditional method can be completed in one step., the operation is simple and easy, which effectively streamlines the process, reduces risk of sample loss and cross contamination, and makes it easy to automate the whole process.
Brief description of drawings
Fig1: Fragment distribution of L1.
Fig 2: Fragment distribution of L2.
Fig 3: Fragment distribution of L3.
Fig 4: Fragment distribution of L4.
Fig 5: Distribution of library construction fragments of L1.
Fig 6: Distribution of library construction fragments of L2.
Fig 7: Distribution of library construction fragments of L3.
Fig 8: Distribution of library construction fragments of L3.
Fig 9: Gene expression of Ll, L2, L3, and L4.
One embodiment of the invention is to combine enzymes, dNTPs, oligos, and buffers required for cell lysis, reverse transcription, template switching, PCR amplification in one reaction mix. Once a single cell, or limited number of cells, is added to the reaction, cell can be first lyzed, the RNA released and reverse transcribed to cDNA, and a sequence tag is added to the 3’ of cDNA by template switching. The cDNA is then amplified in PCR. Different enzymatic reactions can be performed in the same master mix at different temperatures.
We used an RT-PCR one-step master mix (QIAGEN) together with primers needed for template switching and subsequent PCR amplification for proof of principle of the technology. As control, we use a conventional SMART amplification protocol.
I. two steps of the Conventional technology
1 ) Experimental Materials: 100 cells (We used the cell from K562 cell line)
2) Reagent preparation:
Phanta Max Super-Fidelity DNA Polymerase (Vazyme P505-d1)
VAHTS DNA Clean Beads (Vazyme N411-01/02/03)
RNase Inhibitor (Themo Fisher N8080119)
primer
5) Experimental procedure
(1) Configure the PCR mix according to the following system and mix thoroughly:
Reagent | Vol (μL) | Stock |
K562 cells | ||
1 | 10 ^5/μL | |
OligodT30 | 3 | 4 μM |
Nuclease- |
5 | |
Total Volume | 9 |
(2) Perform the reaction on the PCR instrument
Lid Temperature | Reaction VolμMe | Run Time | |
82℃ | 9 μL | ||
| Temperature | Time | |
1 | 72℃ | 0: 03: 00 | |
2 | 72℃→42℃ | 0.1℃/sec | |
3 | 42℃ | 2min |
(3) Configure the Mastter mix according to the following system and mix thoroughly:
4, Incubate the Master Mix at 42 ℃ for 1 min with the PCR instrument to reach the reaction temperature, and mix with the PCR mix,
|
Reaction VolμMe | Run Time |
42 ℃ | 20 μL | 1: 30: 00 |
step 2 | Reaction VolμMe | Run Time |
Lid Temperature | 82℃ | |
72℃ | 20 μL | 0: 10: 00 |
8, PCR amplification after the reverse transcription reaction: Divide the above 20μl product into 2 centrifuge tubes, each 10μl, prepare the following system.
Stock | Vol (μL) | Stock Conc |
Reverse transcription product | 10μl | |
DNA polymerase | 1μl | |
PCR Buffer | 10μl | 5X |
dNTPs | 1μl (10mM) | 10mM |
primer SP1 | 1μl | 100μM |
H2O | Make up 50μl |
Perform the reaction on the PCR instrument
6 Once PCR is completed , the amplified products were purified by 1X magnetic beads
1) . Add 50μl of VAHTS DNA Clean Beads magnetic beads to each tube, shake gently at level 2 for 5min, place on a magnetic stand, let stand until the liquid is clear
2) . Discard the supernatant, add 200 μl of 80%ethanol, stand for 30 s, discard the supernatant and repeat once;
3) . Add 17μl NF-water, resuspend the magnetic beads, mix well, let stand for 5min, put on a magnetic stand until the liquid is clear;
4) . Take 16μl in a new EP tube as a sample.
7 Carry out the library inspection and start the computer.
II. RT-PCR one step method
1) Experimental material: 100 cells ( (We used cells from K562 cell line) )
2) Experimental reagent:
VAHTS DNA Clean Beads (Vazyme N411-01 /02/03)
QIAGEN OneStep RT-PCR Kit (Cat No. /ID: 210210)
RNase Inhibitor (Themo Fisher N8080119)
Primers:
Experimental steps:
(1) Configure the reaction system and mix thoroughly
(2) Place the system in a PCR instrument and perform the reaction as follows.
(3) After the reaction, the amplified products were purified by 1X magnetic beads
1) . Add 50μl of VAHTS DNA Clean Beads magnetic beads to each tube, shake gently at level 2 for 5min, place on a magnetic stand, let stand until the liquid is clear
2) . Discard the supernatant, add 200 μl of 80%ethanol, stand for 30 s, discard the supernatant and repeat once;
3) . Add 17μl NF-water, resuspend the magnetic beads, mix well, let stand for 5min, put on a magnetic stand until the liquid is clear;
4) . Take 16μl in a new EP tube as a sample.
7 Carry out the library inspection and start the computer.
Result
The present invention has been verified by Confirmatory experiment which have proved the feasibility of the method. Now we list the results of a comparative experiment we have done. Although the one-step method is less efficient than the conventional method, it does not affect the later library construction experiment.
Table 1
Reference
[1] Picelli S , Asa K
Faridani O R , et al. Improved Smart-Seq for sensitive full-length transcriptome profiling in single cells [J] . Nature Methods, 2013, 10 (11) .
[2] Faye O, Faye O, Dupressoir A, et al. One-step RT-PCR for detection of Zika virus [J] . Journal of Clinical Virology, 2008, 43 (1) : 96-10
[3] Picelli S , Faridani O R , Bj? Rklund ? K , et al. Full-length RNA-seq from single cells using Smart-seq2 [J] . Nature Protocols, 2014, 9 (1) : 171-181.
[4] Ziegenhain C , Vieth B , Parekh S , et al. Comparative Analysis of Single-Cell RNA Sequencing Methods [J] . Molecular Cell, 2017, 65 (4) : 631-643. e4.
Claims (5)
- A method that can turn minimal amount of RNA or cells to full length cDNA library in a convenient one-tube reaction, comprising:a) method for performing cells lysis in this invention by the temperature;b) reverse transcribe the RNA to cDNA with the oligo-dT primer and reverse transcriptase;c) amplify cDNA.
- The method of Claim 1, wherein the reaction mix contains at least the following components: oligo_dT, reverse transcriptase, Hot start DNA polymerase, dNTP, RNase inhibitor etc.
- The method of Claim 2, whereina) the enzyme in the one-step method can be a mixed enzyme of reverse transcriptase and amplifying enzyme, or it can be a single enzyme enzyme that complete one-step reverse transcription and amplification from mRNA to DNA,b) DNA polymerase can be Hot start DNA polymerase, ordinary DNA polymerase or other enzymes that can complete DNA amplification.
- A method of RNA reverse transcription and cDNA amplification in one system, whereina) system refers to a PCR tube;b) system refers to microfluidic chip, etc.
- A product or kit that includes reagents and automated instrument needed to enable the process as described in Claim 1.
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