CN108342458B - Malt leaching rate evaluation method based on gene expression monitoring - Google Patents
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Abstract
The invention provides a malt extraction rate evaluation method based on gene expression monitoring, which relates to the field of food detection.
Description
Technical Field
The invention relates to the field of food detection, in particular to a malt leaching rate evaluation method based on gene expression monitoring.
Background
The beer contains various components derived from malt, hops, and yeast. The quality of malt directly affects the quality of beer. The malt provides nutrient substances such as sugar, amino acid and the like for yeast fermentation, provides biomacromolecule frameworks such as protein and polysaccharide for beer foam, and simultaneously increases the mellow and smooth taste of the beer. The malt plays a significant role in the production, quality, cost and flavor stability of beer. The selection of high-quality malt becomes the key for producing high-quality beer for beer production enterprises.
There are many indexes for evaluating the quality of malt, and the extraction rate is a key index. The extraction rate is the percentage of dry matter extracted from malt and dissolved in water to dry matter of malt, and the extraction rate and yield are high. It includes the soluble substances of malt itself and the soluble substances formed by the action of enzyme during saccharification, so that the extraction rate reflects the degree of malt dissolution and the quantity of formed enzyme during the malt preparation process of barley, and also reflects the characteristic of how much dry matter is lost after the barley is made into dry malt.
The extraction rate is measured by measuring the sugar degree, density, etc. of the resulting wort by a standard protocol saccharification method and then converting the measured value into the extraction rate. The method has hysteresis and cannot make judgment in advance in the barley germination stage. In addition, the method is complex and time-consuming and labor-consuming to operate. If a more rapid prediction method can be established, the leaching rate of the malt can be predicted from the initial stage of barley germination, a basis can be provided for the adjustment of the malting process, and the production can be rapidly guided.
Tracing back to the source, the difference of the malt extraction rate is caused by the difference of starch and proteolytic enzyme in the malting process. The dissolution of endosperm includes the action of the produced protease on protein between endosperm cell walls, the degradation of cell walls by glucanase, pentosanase and hemicellulase to form porous structure, the entry of amylase and protease into endosperm cells, and a series of degradation reactions to loose endosperm tissue and expose starch to form powdered structure. As can be seen from the dissolution process, enzymes play a very important role in the malting process. The type and activity of these hydrolases directly determine the solubilization status of the endosperm and thus the quality of the malt. The activity of these hydrolases is determined by the expression level of the relevant genes in the malting process. At present, there is no method for evaluating the leaching rate based on these hydrolase genes.
Disclosure of Invention
The invention aims to provide a malt leaching rate evaluation method based on gene expression monitoring, which is used for evaluating the malt leaching rate by establishing a method for predicting the malt leaching rate based on the gene expression monitoring of starch and proteolytic enzyme; compared with the traditional determination method, the method has the advantages of high accuracy, high efficiency and simplicity.
In order to achieve the above object, the present invention provides a malt extraction rate evaluation method based on gene expression monitoring, comprising extraction and purification of total RNA, preparation of cDNA template by reverse transcription reaction, multiple PCR reaction, obtaining gene electrophoresis pattern by capillary electrophoresis, obtaining expression peak height H of malt hydrolysis related gene by pattern data analysis, calculating malt extraction rate by using the expression peak height H of malt hydrolysis related gene,
aiming at the malt hydrolysis related genes L D, AMY1, AMY4, G L U, ASP, CYS, SERI, SERIII, MET, TRX, PDI, SEP, WRKY and internal control gene ACT;
preparing a cDNA template by reverse transcription reaction, wherein the application primers are SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 and SEQ ID NO. 28;
primers SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.27 are applied in a multiplex PCR reaction;
wherein, the formula for calculating the malt extraction rate by using the expression peak height H of the malt hydrolysis related gene is as follows:
malt extraction rate 82.864+10 [ [0.233 ] H (AMY1)/H (act) -0.272 ] H (trx)/H (act) -0.207 ] H (SER i)/H (act) -0.381 ] H (met)/(act) -0.195 ] H (cys)/H (act) -0.061H (pdi)/H (act) -0.182 ] H (asp)/H (act) -0.009 ] H (sep)/H (act) -0.122 ] H (wrpdi)/H (act) -0.970H (L D)/ac t-2.094) H (AMY 57)/H (act) -0.088 ] G L H (act)/09h (act) H (act) -0.09h (act).
The specific malt extraction rate evaluation method comprises the following steps:
(1) malt extraction prediction was performed using the following multiplex primers:
screening genes and internal control genes related to barley germination process protein and polysaccharide hydrolysis, and designing specific upstream and downstream primers which accord with GeXP multiple PCR reaction characteristics and are expressed by the genes related to barley germination process protein and polysaccharide hydrolysis, and referring to Table 1.
TABLE 1 specific upstream and downstream primers for malt leaching rate-related genes
Wherein each primer is prepared into 100 mu M storage solution respectively, and mixed in equal proportion to prepare the mixed primer working solution. The working solution of the downstream mixed primer is 500nM, and the working solution of the upstream mixed primer is 200 nM;
(2) total RNA extraction and purification: extracting purified malt total RNA from malt cells of barley by adopting a general RNA extraction and purification method;
(3) preparation of cDNA template by reverse transcription reaction A first strand cDNA was synthesized using malt total RNA as a template, using Genome L ab from Beckmann CoulterTMThe GeXP boot kit takes the downstream primer of the multiple primers in the step (1) as a specific primer, and the reaction system is 10 mu L, which is shown in Table 2.
TABLE 2 reaction System for preparation of cDNA template by reverse transcription reaction
| Composition per tube | Standard reaction |
| DNase/RNase-free water | 3μL |
| 5 × reverse transcription buffer solution | 4μL |
| KANrRNA (1:50 dilution) | 5μL |
| Reverse transcriptase | 1μL |
| Downstream primer (500nM) | 2μL |
| RNA template (5-20 ng/. mu. L) | 5μL |
The reaction parameters for preparing cDNA template by reverse transcription reaction are set as follows:
1 minute at 48 ℃; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.
(4) Performing multiplex PCR by using cDNA template prepared by the synthesized reverse transcription reaction as template and upstream primer of the multiplex primer in (1) as specific primer, setting 3 parallel tubes for each sample, and adopting DNA polymerase of BeckmanCoulte company and Genome L abTMGeXP start kit, the specific reaction conditions are shown in Table 3.
TABLE 3 reaction conditions for multiplex PCR
The RT-PCR amplification parameters were set as follows:
10 minutes at 95 ℃, 30 seconds at 94 ℃, 30 seconds at 56 ℃ and 1 minute at 71 ℃; 35 cycles.
(5) And (3) performing capillary electrophoresis on multiple PCR products, namely adding 1 mu L PCR multiple products into holes of an upper sample plate filled with a mixture of 39 mu L of 95% deionized formamide (S L S) and 400bp Marker, uniformly mixing the mixture by using a pipette, covering a drop of paraffin oil, adding 250 mu L of separation buffer solution into each hole of a buffer solution plate, and performing capillary electrophoresis on a machine to obtain a capillary electrophoresis pattern after all the products are prepared.
(6) Analyzing the capillary electrophoresis pattern to obtain the expression peak height H of the malt hydrolysis related gene.
(7) Calculating the malt leaching rate by using the expression peak height H of the malt hydrolysis related gene, wherein the formula is as follows:
malt extraction rate 82.864+10 [ [0.233 ] H (AMY1)/H (act) -0.272 ] H (trx)/H (act) -0.207 ] H (SER i)/H (act) -0.381 ] H (met)/(act) -0.195 ] H (cys)/H (act) -0.061H (pdi)/H (act) -0.182 ] H (asp)/H (act) -0.009 ] H (sep)/H (act) -0.122 ] H (wrpdi)/H (act) -0.970H (L D)/ac t-2.094) H (AMY 57)/H (act) -0.088 ] G L H (act)/09h (act) H (act) -0.09h (act).
Compared with the prior art, the invention has the advantages and positive effects that:
(1) the leaching rate can be quickly and accurately evaluated by adopting the evaluation method.
(2) Compared with the conventional gene expression quantitative analysis technology (fluorescent quantitative PCR), the GeXP multiple gene expression quantitative analysis technology adopted by the invention has the characteristics of stronger specificity and sensitivity, high automation degree and the like, and ensures the reliability and repeatability of results. In addition, the GeXP multiple gene expression quantitative analysis technology can simultaneously detect the expression abundance of up to 30 genes, thereby greatly shortening the experimental time.
(3) The invention can prejudge the leaching rate of the malt in advance, and can lead the prediction time to be 3-4 days in advance compared with the traditional method of detecting the leaching rate after obtaining the malt, thereby being capable of guiding a factory to carry out process adjustment.
Drawings
FIG. 1 is a diagram of capillary electrophoresis provided in example 1 of the present invention;
FIG. 2 is a diagram of capillary electrophoresis provided in example 2 of the present invention;
FIG. 3 is a diagram of capillary electrophoresis provided in example 3 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment of the invention provides a Canadian malt leaching rate evaluation method based on gene expression monitoring.
The method comprises extracting total RNA, preparing cDNA template by reverse transcription reaction, performing multiple PCR reactions, performing capillary electrophoresis to obtain gene electrophoresis pattern, analyzing pattern data to obtain expression peak height H of malt hydrolysis related gene, calculating malt leaching rate by using the expression peak height H of malt hydrolysis related gene,
aiming at the malt hydrolysis related genes L D, AMY1, AMY4, G L U, ASP, CYS, SERI, SERIII, MET, TRX, PDI, SEP, WRKY and internal control gene ACT;
preparing a cDNA template by reverse transcription reaction, wherein the application primers are SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 and SEQ ID NO. 28;
primers SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.27 were used in the multiplex PCR reaction.
(1) According to L D, AMY1, AMY4, G L U, ASP, CYS, SER I, SER III, MET, TRX, PDI, SEP, WRKY and ACT gene full-length sequences, Primer Premier 5 software is used for designing specific upstream and downstream primers which are in accordance with the GeXP multiplex PCR reaction characteristics and are related to barley germination process protein and polysaccharide hydrolysis, and the Primer sequences are shown in a table 4.
TABLE 4 specific upstream and downstream primers for malt leaching rate-related genes
(2) In the process of malting, taking green malt germinating for 1 day, grinding and crushing by liquid nitrogen to obtain a malt sample, and immediately extracting RNA from the malt sample or storing the malt sample at-80 ℃.
(3) Extraction of total RNA by TRIZO L method
1) Taking 50-100mg of sample, and adding 1ml of Trizol;
2) after shaking and cracking, centrifuging at 12000g at 4 ℃ for 5min, transferring the supernatant to a Phasemarker tube, and standing for 5 min;
3) adding 0.2ml chloroform, manually shaking for 15s, and standing for 10 min; centrifuging at 12000g at 4 deg.C for 10 min;
4) taking 550 μ l of supernatant fluid of about 450-;
5) adsorbing the mixed solution by silica gel, centrifuging for 1min at 12000g, and removing the flowing-down waste liquid;
6) add 700. mu.l of WB1(Thermo Fisher RNA purification kit: GeneJET Plant RNAtreatment Kit), centrifuging at 12000g for 1min, and removing the flowing-down waste liquid;
7) add 500. mu.l of WB2(Thermo Fisher RNA purification kit: GeneJET Plant RNAtreatment Kit), centrifuging at 12000g for 1min, and removing the flowing-down waste liquid;
8) add 500. mu.l of WB2(Thermo Fisher RNA purification kit: GeneJET Plant RNAtreatment Kit), centrifuging at 12000g for 1min, and transferring the silicone tube to a 1.5ml EP tube;
9) adding 50 μ l of clean-free water, standing for 1min, and centrifuging at 12000g for 1 min;
10) the centrifuged RNA solution was collected and stored at-80 ℃.
(4) Preparing a cDNA template by reverse transcription reaction:
using the above RNA as a template, Genome L ab from Beckman Coulter was usedTMGeXP start kit, the downstream primer of the multiplex primer in Table 5 above is the specific primer, the first strand of cDNA is synthesized, the reaction system is 10 μ L.
The cDNA first strand synthesis reaction system setup is shown in Table 5, in which NTC and RT-Negative control:
TABLE 5 reaction conditions for reverse transcription to prepare cDNA templates
| Composition per tube | NTC | RT- | Standard reaction | |
| DNase/RNase-free water | 8μL | 4μL | 3μL | |
| 5 × reverse transcription buffer solution | 4μL | 4μL | 4μL | |
| KANrRNA (1:50 dilution) | 5μL | 5μL | 5μL | |
| | 1μL | 0 | 1μL | |
| Downstream primer (500nM) | 2μL | 2μL | 2μL | |
| RNA template (5-20 ng/. mu. L) | 0 | 5μL | 5μL |
The reaction parameters for preparing cDNA template by reverse transcription reaction are set as follows:
1 minute at 48 ℃; 60 minutes at 42 ℃; 95 ℃ for 5 minutes.
(5)RT-PCR：
DNA polymerase and Genome L ab from Beckman Coulter were usedTMGeXP start kit. RT-PCR amplification reaction was performed using cDNA templates prepared by reverse transcription reaction synthesized in the above Table 2 as templates and the upstream primers of the multiplex primers in the above Table 5 as specific primers, and 3 parallel channels were provided for each sample, and the specific reaction conditions are shown in Table 6.
TABLE 6 reaction conditions for RT-PCR
| Composition (I) | Volume (mu L) |
| 25mM MgCl2 | 4.0μL |
| 5 × PCR buffer | 4.0μL |
| DNA polymerase | 0.7μL |
| Upstream primer (200nM) | 2μL |
| First strand cDNA | 9.3μL |
The RT-PCR amplification parameters were set as follows:
10 minutes at 95 ℃; 30 seconds at 94 ℃; 56 ℃ for 30 seconds; 71 ℃ for 1 minute (35 cycles).
(6) Capillary electrophoresis of multiple PCR products, namely adding 1 mu L PCR multiple products into the holes of an upper sample plate respectively filled with 39 mu L of 95% deionized formamide (S L S) and 400bpMarker mixed solution, uniformly mixing the mixture by using a pipette, covering a drop of paraffin oil, adding 250 mu L of separation buffer solution into each hole of a buffer solution plate, and performing capillary electrophoresis on a machine after all the products are prepared to obtain a capillary electrophoresis pattern, wherein the pattern is shown in figure 1.
(7) Analysis of capillary electrophoresis patterns
Analyzing the capillary electrophoresis result by using GeXP system parameters, recording the result, as shown in figure 1, measuring the expression peak height H of the malt hydrolysis related gene, wherein each peak with different sizes corresponds to the corresponding gene, and the height of each peak corresponds to the expression amount of the gene, and the result is shown in the following table 7.
TABLE 7 Peak height H of malt leaching Rate-related genes of Canada
(8) Calculating the malt leaching rate by using the expression peak height H of the malt hydrolysis related gene, wherein the formula is as follows:
malt extraction rate 82.864+10 [ [0.233 ] H (AMY1)/H (ACT) -0.272 ] H (TRX)/H (ACT) -0.207 ] H (SER I)/H (ACT)) +0.381 ] H (MET)/H (ACT)) +
0.195*H(CYS)/H(ACT)+0.061*H(PDI)/H(ACT)-0.182*H(ASP)/H(ACT)+0.009*H(SEP)/H(ACT)-0.122*H(WRKY)/H(ACT)-0.970*H(LD)/H(AC T)-2.094*H(AMY4)/H(ACT)-0.088*H(GLU)/H(ACT)+0.097*H(SERⅢ)/H(ACT)]。
The calculated malt extraction rate is 81.6 percent, and compared with the extraction rate of 80.3 percent obtained by the traditional pycnometer method, the extraction rates of the two methods are similar, which shows that the malt extraction rate evaluation method based on gene expression monitoring has high accuracy and efficiency.
Example 2
The embodiment of the invention provides an Australian malt leaching rate evaluation method based on gene expression monitoring.
Steps (1) to (6) were the same as in example 1, and the capillary electrophoresis pattern was as shown in FIG. 2.
(7) Analysis of capillary electrophoresis patterns
Analyzing the capillary electrophoresis result by using GeXP system parameters, recording the result, as shown in figure 2, measuring the expression peak height H of the malt hydrolysis related gene, wherein each peak with different sizes corresponds to the corresponding gene, and the height of each peak corresponds to the expression amount of the gene, and the result is shown in the following table 8.
TABLE 8 Australian malt extraction Rate related Gene Peak height H
(8) Calculating the malt leaching rate by using the expression peak height H of the malt hydrolysis related gene, wherein the formula is as follows:
malt extraction rate 82.864+10 [ [0.233 ] H (AMY1)/H (act) -0.272 ] H (trx)/H (act) -0.207 ] H (SER i)/H (act) -0.381 ] H (met)/(act) -0.195 ] H (cys)/H (act) -0.061H (pdi)/H (act) -0.182 ] H (asp)/H (act) -0.009 ] H (sep)/H (act) -0.122 ] H (wrpdi)/H (act) -0.970H (L D)/ac t-2.094) H (AMY 57)/H (act) -0.088 ] G L H (act)/09h (act) H (act) -0.09h (act).
The calculated malt extraction rate is 79.8 percent, and is 79.4 percent compared with the extraction rate obtained by the traditional pycnometer method, and the extraction rates of the two are very close, which indicates that the malt extraction rate evaluation method based on gene expression monitoring has high accuracy and efficiency.
Example 3
The embodiment of the invention provides a domestic malt leaching rate evaluation method based on gene expression monitoring.
Steps (1) to (6) were the same as in example 1, and the capillary electrophoresis pattern was as shown in FIG. 3.
(7) Analysis of capillary electrophoresis patterns
Analyzing the capillary electrophoresis result by using GeXP system parameters, recording the result, as shown in figure 3, measuring the expression peak height H of the malt hydrolysis related gene, wherein each peak with different sizes corresponds to the corresponding gene, and the height of each peak corresponds to the expression amount of the gene, and the result is shown in the following table 9.
TABLE 9 Peak height H of gene related to the Leaching Rate of domestic malt
| Serial number | Name of Gene | Peak height H |
| 1 | AMY1 | 42135 |
| 2 | TRX | 98401 |
| 3 | SERⅠ | 103497 |
| 4 | MET | 31957 |
| 5 | CYS | 134743.5 |
| 6 | PDI | 166582.5 |
| 7 | ASP | 141657 |
| 8 | ACT | 109440 |
| 9 | SEP | 141243 |
| 10 | WRK | 23684.5 |
| 11 | LD | 27134 |
| 12 | AMY4 | 3538 |
| 13 | GLU | 27011 |
| 14 | SERⅢ | 63887 |
(8) Calculating the malt leaching rate by using the expression peak height H of the malt hydrolysis related gene, wherein the formula is as follows:
malt extraction rate 82.864+10 [ [0.233 ] H (AMY1)/H (act) -0.272 ] H (trx)/H (act) -0.207 ] H (SER i)/H (act) -0.381 ] H (met)/(act) -0.195 ] H (cys)/H (act) -0.061H (pdi)/H (act) -0.182 ] H (asp)/H (act) -0.009 ] H (sep)/H (act) -0.122 ] H (wrpdi)/H (act) -0.970H (L D)/ac t-2.094) H (AMY 57)/H (act) -0.088 ] G L H (act)/09h (act) H (act) -0.09h (act).
The calculated malt extraction rate is 78.6 percent, and is 78.9 percent compared with the extraction rate obtained by the traditional pycnometer method, and the extraction rates of the two are close, which shows that the malt extraction rate evaluation method based on gene expression monitoring has high accuracy and efficiency.
Sequence listing
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Claims (3)
1. A malt extraction rate evaluation method based on gene expression monitoring is characterized by comprising the steps of extracting and purifying total RNA, preparing a cDNA template through reverse transcription reaction, performing multiple PCR reaction, obtaining a gene electrophoresis pattern through capillary electrophoresis, obtaining an expression peak height H of a malt hydrolysis related gene through pattern data analysis, calculating the malt extraction rate by utilizing the expression peak height H of the malt hydrolysis related gene,
aiming at the malt hydrolysis related genes L D, AMY1, AMY4, G L U, ASP, CYS, SERI, SERIII, MET, TRX, PDI, SEP, WRKY and internal control gene ACT;
preparing a cDNA template by reverse transcription reaction, applying primers of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 and SEQ ID NO.28, and synthesizing a first cDNA chain;
carrying out multiplex PCR reaction by taking the first cDNA chain as a template, wherein primers used in the multiplex PCR reaction are SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO. 27;
wherein, the formula for calculating the malt extraction rate by using the expression peak height H of the malt hydrolysis related gene is as follows:
malt extract rate =82.864+10 [ [0.233 ] H (AMY1)/H (ACT) -0.272 ] H (TRX)/H (ACT) -0.207 [. H (SER I)/H (ACT)) +0.381 ] H (MET)/H (ACT)) +
0.195*H(CYS)/H(ACT)+0.061*H(PDI)/H(ACT)-0.182*H(ASP)/H(ACT)+0.009*H(SEP)/H(ACT)-0.122*H(WRKY)/H(ACT)-0.970*H(LD)/H(ACT)-2.094*H(AMY4)/H(ACT)-0.088*H(GLU)/H(ACT)+0.097*H(SERⅢ)/H(ACT)]。
2. The method of claim 1, wherein the total reaction system of the multiplex PCR reaction is 20 μ L, wherein the total reaction system is 25mM MgCl24.0 mu L, 5 × PCR buffer solution 4.0 mu L, DNA polymerase 0.7 mu L, cDNA template application primer 2 mu L for reverse transcription reaction, and primer 9.3 mu L for multiplex PCR reaction, wherein the amplification parameters comprise pre-denaturation at 95 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 71 ℃ for 1min, and circulation is carried out for 35 times.
3. The method for evaluating a malt extraction ratio based on gene expression monitoring according to any one of claims 1 to 2, wherein the size of the product of the malt hydrolysis-related gene L D is 290bp, the size of the product of AMY1 is 148bp, the size of the product of AMY4 is 339bp, the size of the product of G L U is 374bp, the size of the product of ASP is 222bp, the size of the product of CYS is 208bp, the size of the product of SERI is 180bp, the size of the product of SERIII is 395bp, the size of the product of MET is 197bp, the size of the product of TRX is 176bp, the size of the product of PDI is 218bp, the size of the product of SEP is 268bp, the size of the product of KYWR is 283bp, and the size of the product of the internal control gene ACT is 237 bp.
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