How to improve the efficiency of LncRNA reverse transcription?

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Long non-coding RNA, lncRNA is a non-coding RNA with a length greater than 200 nucleotides, generally between 200-100000 nt. lncRNA regulates gene expression at epigenetic, transcription and post-transcriptional levels, and participates in X chromosome silencing, genome imprinting and chromatin modification, transcription activation, transcription interference, nuclear transport, cell cycle regulation, cell differentiation regulation, and dose compensation effects (Dosage compensation effect) and many other important regulation processes are closely related to the occurrence, development and prevention of human diseases, and are currently one of the hot spots in the field of biomedicine.

01 Types and characteristics of LncRNA

lncRNA is divided into many types according to the different sources of its formation, as shown in the figure above. Has the following characteristics:

1. lncRNAs are usually longer, with dynamic expression and different splicing methods during differentiation
2. Compared with coding genes, lncRNA usually has a lower expression level
3. Most lncRNAs have obvious temporal and spatial expression specificity in the process of tissue differentiation and development
4. There are characteristic expressions in tumors and other diseases
5. The subcellular locations of lncRNA are diverse
6. The sequence is low in conservation, but the function has a certain degree of conservation, etc.

02 Frequently Asked Questions about LncRNA Reverse Transcription

Due to the low content of lncRNA in cells, long length and high-level structure, such RNA often has complex structures such as stem loops or hairpins, and RT-qPCR is prone to problems of unsuccessful reverse transcription or low efficiency.

03 LncRNA reverse transcription solution

In the central rule, the process of RNA viruses using their own reverse transcriptase to synthesize DNA using RNA as a template is called reverse transcription. The process of using RNA virus reverse transcriptase to synthesize cDNA template in vitro is called reverse transcription. The main elements included in the reverse transcription system: RNA template, reverse transcriptase, primers and other components.

1.RNA template

The influence of RNA template on reverse transcription is reflected in its structure, purity and integrity. The higher the purity and integrity of the extracted RNA, the higher the efficiency of reverse transcription and the more accurate the subsequent quantitative results. The RNA purified by commonly used RNA extraction reagents often contains residues of alcohol and guanidine salts, which have a strong inhibitory effect on most reverse transcriptase.

2.Reverse transcriptase

Reverse transcriptase has a key influence in the entire reverse transcription system. The suitable temperature for the reverse transcriptase used in most laboratories is generally 42°C. In fact, the opening of the high-level RNA structure requires a higher temperature, and the requirements for the reverse transcriptase are higher.

3.Primer

Reverse transcription primers include gene-specific primers, random primers and Oligo dT primers. Most lncRNAs do not contain polyA tails, and it is important to properly match the random primers with Oligo dT.

4.Other components

In view of the fact that RNA is particularly easy to degrade, the introduction of components in the reverse transcription system can be minimized, which can effectively prevent the entry of RNase and ensure the smooth progress of reverse transcription.

Foregene obtains from RNA template, reverse transcription to qPCR Comprehensively escort your lncRNA detection

Animal Total RNA Isolation Kit (with gDNA removal column)

Cell Total RNA Isolation Kit (with gDNA removal column)

Lnc-RT HeroTM I(With gDNase)(Super Premix for first-strand cDNA synthesis from lncRNA)

Real Time PCR EasyTM-SYBR Green I

Advantages of RNA extraction:

The kit uses the third-generation RNA extraction technology, no need to use DNase.

11 minutes from 96, 24, 12, and 6-well plate cultured cells, high-efficiency extraction of gDNA-free, high-purity, high-quality total cell RNA.

Advantages of lncRNA reverse transcription:

A reverse transcription system specially developed for LncRNA to quickly remove genomic DNA contamination.

The reverse transcription system has high thermal stability and still has good reverse transcription performance at 50°C.

Templates with high GC content and complex secondary structure can be reversed with high efficiency.

Advantages of qPCR:

Hot start Foregene Taq Polymerase has higher amplification efficiency, higher amplification sensitivity, and higher amplification specificity.

The optimized Real PCR Easy Mix makes SYBR Green I have higher detection sensitivity, and its fluorescence intensity is 3-5 times that of similar products, which can meet the needs of different types of fluorescence quantitative experiments.