• facebook
  • linkedin
  • youtube

RT-qPCR is the basic experiment of molecular biology, and everyone must be familiar with it. It mainly includes three steps: RNA extraction, reverse transcription into cDNA, and real-time fluorescent quantitative PCR. It doesn’t help, what is going on? It is likely that there is a problem with the reverse transcription experiment ! Although it seems that the reverse transcription experiment only needs to add RNA, dNTP, primers, and reverse transcriptase to the centrifuge tube and mix well, but in the actual operation process, there are still many details that need to be paid attention to. Let’s learn about it!

How to judge the quality of RNA?
To obtain cDNA, the quality of RNA is critical! RNA quality can be detected mainly from two aspects:
(1) RNA integrity: RNA integrity can be verified by agarose gel electrophoresis . Taking eukaryotes as an example, the complete total RNA has three clear bands, the molecular weights from large to small are 28S, 18S, and 5S, and 28S is twice as bright as 18S; if three bands can be seen, but the band type is blurred or Diffusion means that the RNA is partially degraded. At this time, please perform the reverse transcription reaction immediately and increase the template input appropriately; if only a band with a small molecular weight or no band can be seen, the RNA has been completely degraded and needs to be re-extracted . Agilent 2100 indicates the integrity of RNA with peak diagram and RIN value. If the nucleic acid is intact, the baseline of the electropherogram is flat; if the nucleic acid is severely degraded, the baseline is uneven and more degradation peaks appear; the value of RIN reflects the integrity of the RNA, within the range of 0-10, the larger the value, the better the quality of the RNA. Well, the higher the degree of completeness.
(2) Purity of RNA: The ratio of OD260/280 can be detected by UV spectrophotometry . If the ratio of OD260/280 is between 1.9 and 2.1, the purity is very good.
Residual genomic DNA can lead to inaccurate quantitative results
When RNA is extracted, the RNA we get may be mixed with genomic DNA (gDNA) that has not been cleaned up. Therefore, the cDNA after reverse transcription will also be mixed with gDNA . During the downstream qPCR reaction, cDNA and gDNA may be amplified simultaneously, resulting in a relatively small CT value, so the results may be biased.
So what should we do in this situation? Foregene suggests:
(1) Perform genome cleaning on the reversed RNA, which can be removed by column extraction during RNA extraction;
(2) Treat the extracted RNA with DNase I , but terminate it with EDTA;
of reverse transcription reagents with genome clearing modules ;

How to choose primers for reverse transcription?
Reverse transcription primers also affects the outcome of the reverse transcription reaction. You can choose random primers, Oligo dT or gene-specific primers for reverse transcription according to the specific circumstances of the experiment:
(1) Specific transcripts : gene-specific primers are recommended;
(2) Long fragment transcripts : Oligo dT/gene-specific primers are recommended;
(3) Internal fragments of long-segment transcripts : gene-specific primers/ random primers /random primers + Oligo dT. If the subsequent qPCR experiment is performed, Oligo dT cannot be used alone, because using Oligo dT alone may cause 3′ end bias , leading to inaccurate qPCR experiment results;
(4) miRNA : Stem-loop primers or tailing primers can be used.

How many times should the reverse transcription product cDNA be diluted for quantification?
After obtaining the cDNA of the reverse transcription product, how many times the cDNA should be diluted for qPCR experiments is very important. If the cDNA concentration is too high or too low, the amplification efficiency may be affected. Can the cDNA concentration be measured, and how should it be done?
(1) The cDNA concentration of the reverse transcription product cannot be measured, because in addition to the cDNA product, the reverse transcription product also contains reverse transcription residual Buffer, reverse transcriptase, primers, etc., which will interfere with the concentration measurement results and cause OD260/280, OD260/ 230  ratio abnormal and therefore does not reflect true cDNA yield. At this time, some friends will say, then I will measure the concentration after purification; here,Foregene would like to remind that the cDNA is not recommended to be purified, because the length of the cDNA obtained by the reversal is different, and the short cDNA will be lost in the purification.
(2) So what to do? Before the qPCR experiment, the dilution gradient of the cDNA can be determined through the pre-experiment . For example: use cDNA stock solution, 10-fold dilution, and 100-fold dilution as templates for qPCR experiments, and select the dilution factor with a CT value in the range of 18-28.

How should miRNAs be reverse transcribed?
miRNA is a single-stranded small molecule RNA with a size of about 22 nt that does not code for protein. Because of its short length, conventional qPCR method is difficult to directly quantify it, so it is often necessary to extend miRNA; the commonly used reverse transcription methods for miRNA include stem-loop method and tailing method.
The stem-loop method is to extend the miRNA by adding stem-loop primers. This detection method has higher sensitivity and specificity, but the detection throughput is low. One reverse transcription can only detect one miRNA and an internal reference; the tail-adding method is composed of two It is completed by the joint action of two enzymes, which are PolyA polymerase and reverse transcriptase. PolyA polymerase is responsible for adding PolyA tails to miRNA to increase its length, and reverse transcriptase performs reverse transcription reaction. This method has high detection throughput and can detect multiple miRNAs and internal references in one reverse transcription, but the sensitivity and specificity are low in the stem-loop method.


Post time: Feb-17-2023