A-1 RNA is degraded
——Purify high quality RNA with no contamination. The material from which RNA is extracted should be as fresh as possible to prevent RNA degradation. Analyze RNA integrity on denatured gel before RT reaction. After RNA extraction, it should be stored in 100% formamide. If RNase inhibitor is used, the heating temperature should be <45°C, and the pH should be less than 8.0, otherwise the inhibitor will release all bound RNase. Moreover, RNase inhibitor should be added in solutions containing ≥ 0.8 mM DTT.
A-2 RNA contains inhibitors of reverse transcription reactions
——Reverse transcription inhibitors include SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, guanidine salt, etc. Mix the control RNA with the sample, and compare the yield with the control RNA reaction to check whether there is an inhibitor. Wash RNA precipitation with 70% (v/v) ethanol to remove inhibitors.
A-3 Insufficient annealing of primers used for synthesizing the first strand of cDNA
——Determine that the annealing temperature is suitable for the primers used in the experiment. For random hexamers, it is recommended to keep the temperature at 25°C for 10 min before reaching the reaction temperature. For gene-specific primers (GSP), try other GSP, or switch to oligo(dT) or random hexamer.
A-4 Small amount of starting RNA
——Increase the amount of RNA. For RNA samples less than 50 ng, 0.1 μg to 0.5 μg acetyl BSA can be used in the first strand cDNA synthesis
A-5 The target sequence is not expressed in the analyzed tissues.
——Try other tissues.
A-6 PCR reaction fails
——For two-step RT-PCR, the cDNA template in the PCR step cannot exceed 1/5 of the reaction volume.