It is mainly related to the genes, and reverse transcription of long fragment is not feasible for most genes. Firstly, the efficiency of reverse transcription is far lower than that of PCR. Secondly, the GC rich region and secondary structure of many genes restrict both reverse transcription and PCR. Finally, the fidelity and amplification efficiency of PCR are difficult to guarantee at the same time. In the process of reverse transcription, no one can guarantee to get long fragment for low copy genes, especially using oligo dT. As for 5’ UTR with more GC, it is even more difficult. Therefore, it is still a reasonable method to reverse transcript with random primers, find the natural cleavage sites in the target fragment, amplify by segments, and then perform the restriction digestion and ligation. In general, it is difficult to directly amplify fragments larger than 2 kb, but it is not always impossible to obtain:

1.First of all, guarantee the integrity of RNA/mRNA, and TRIZOL extraction is preferred.

2.M-MLV RT-PCR kit can be directly used. Extend annealing time and increase cycle number in the amplification process properly. Alternatively, nested PCR can be applied, or carry out one or two reactions first with appropriately extended denaturation and extension time before normal PCR amplification, which may help to extend fragments. Pay attention to the fidelity of the polymerase.

3.Long Taq can be used in PCR to obtain ideal results.

4.For protein expression application, high fidelity polymerase should be applied.

There are two kinds of reverse transcriptase offered by TIANGEN: Quant/King RTase and TIANScript M-MLV. The main difference between them is input amount of templates. Quant is a unique reverse transcriptase, which is different from the commonly used M-MLV derived from Moloney murine leukemia virus. Quant is a new high-efficiency reverse transcriptase recombinantly expressed by engineering Escherichia coli. Quant is suitable for amplifying 50 ng-2 μg of RNA with high reverse transcriptional activity and high yield. Compared with ordinary MMLV or AMV, Quant’s biggest characteristic is that it has very strong affinity with RNA templates and can reverse transcript complex templates without high temperature denaturation. For templates with higher GC content, the reverse efficiency is higher. However, this reverse transcriptase has RNase H activity, which may affect the length of cDNA product (suitable for < 4.5 kb templates). For conventional reverse transcription, TIANScript MMLV reverse transcriptase is recommended. This RTase is a modified enzyme with very weak RNase H activity, which is suitable for long (> 5 kb) cDNA synthesis.

One-step reverse transcription and PCR amplification are completed in the same tube without opening the tube cover between cDNA synthesis and amplification, which is helpful to reduce contamination. Since all cDNA samples obtained are used for amplification, the sensitivity is higher, with a minimum of 0.01 pg of total RNA. For successful one-step RTPCR, gene-specific primers are generally used to initiate cDNA synthesis. The two-step method, namely reverse transcription and PCR amplification is carried out in two steps. Firstly reverse transcription is carried out from an RNA template to obtain cDNA, and the obtained cDNA is subjected to one or more different PCR reactions. The two-step method can use oligo(dT) or random primers to guide the synthesis of the first strand of cDNA, and can reverse transcribe all mRNA information from a specific sample.