How to improve the sensitivity of RT-PCR reaction system

1. Isolate high-quality RNA

Successful cDNA synthesis comes from high-quality RNA. High-quality RNA should be full-length and free of reverse transcriptase inhibitors such as EDTA or SDS, and minimize genomic DNA contamination. The quality of RNA determines the maximum amount of sequence information you can transcribe to cDNA.

2. Use reverse transcriptase without RNaseH activity

RNase inhibitors are often added in the reverse transcription reaction to increase the length and yield of cDNA synthesis. Both M-MLV and AMV have endogenous RNaseH activity in addition to their own polymerase activity. RNaseH activity and polymerase activity compete with each other for the hybrid strand formed between the RNA template and the DNA primer or cDNA extension strand, and degrade the RNA strand in the RNA:DNA complex. The RNA template degraded by RNaseH activity can no longer be used as an effective substrate for cDNA synthesis, reducing the yield and length of cDNA synthesis. Therefore, eliminating or greatly reducing the RNaseH activity of reverse transcriptase will be of great benefit.

3. Increase the reverse transcription temperature

A higher holding temperature helps to open the RNA secondary structure and increases the yield of the reaction. For most RNA templates, in the absence of buffer or salt, the RNA and primers are incubated at 65°C and then quickly cooled on ice to eliminate most of the secondary structure so that the primers can bind.

4. Additives to promote reverse transcription

Additives including glycerol and DMSO to the *strand synthesis reaction can reduce the stability of the nucleic acid double-strand and untie the RNA secondary structure. You can add 20% glycerol or 10% DMSO without affecting SuperScriptⅡ or M- MLV activity. AMV can also tolerate 20% more glycerol without reducing activity.

5. RNaseH treatment

Treatment of cDNA synthesis reaction with RNaseH before PCR can increase sensitivity. For some templates, the RNA in the cDNA synthesis reaction prevents the binding of the amplified product. In this case, RNaseH treatment can increase sensitivity. Generally, when amplifying longer full-length cDNA target templates, RNaseH treatment is necessary, such as low-copy tuberous scherosis II. For this difficult template, RNaseH processing strengthened the signal generated by SuperScriptⅡ or AMV synthesized cDNA. For most RT-PCR reactions, RNaseH treatment is optional, because the PCR denaturation step incubation at 95°C generally hydrolyzes the RNA in the RNA:DNA complex.

6. Improvements in detection methods for small amounts of RNA

When there is only a small amount of RNA, RT-PCR is particularly challenging. The glycogen as a carrier added during the RNA isolation process helps increase the yield of small samples. You can add RNase-free glycogen at the same time as Trizol. For a sample of 106 cultured cells in tissues less than 50 mg, the recommended concentration of RNase-free glycogen is 250 μg/ml. Adding acetylated BSA to the reverse transcription reaction using SuperScript Ⅱ can increase the sensitivity, and for small amounts of RNA, reducing the amount of SuperScript Ⅱ and adding 40 units of RnaseOut nuclease inhibitor can increase the detection level. If glycogen is used in RNA isolation, it is still recommended to add BSA or RNase inhibitor when using SuperScriptⅡ for reverse transcription reaction.

7. Reduce genomic DNA pollution:

A potential difficulty encountered by RT-PCR is that there is genomic DNA contamination in the RNA. The RNA can be treated with amplification-grade DNase I to remove the contaminated DNA before reverse transcription. The sample was incubated in 2.0 mM EDTA at 65°C for 10 minutes to stop the DNase I digestion. EDTA can chelate magnesium ions and prevent the magnesium ion-dependent DNA hydrolysis that occurs at high temperatures. In order to separate the amplified cDNA from the amplified product of the contaminated genomic DNA, primers that anneal to the separated exons can be designed. PCR products derived from cDNA will be shorter than products derived from contaminated genomic DNA. In addition, a control experiment without reverse transcription is performed on each RNA template to determine whether a given fragment is derived from genomic DNA or cDNA. The PCR product obtained without reverse transcription is derived from the genome.