The principles, parameters and experimental steps of reverse pcR.

Reverse transcription PCR (RT-PCR) principle reverse transcription PCR (RT-PCR) has high sensitivity, simple operation and other advantages, often used in gene quantitative analysis, biological testing, etc. , in addition to the commonly used reverse transcriptite PCR cloning purpose gene.
this paper mainly describes the principle of reverse transcription PCR, important parameters and operational considerations. the synthesis of
cDNA is an important part of RT-PCR.
using mRNA as a template, with the catalyst of reverse transcriptase, random primers, oligo (dT) or gene-specific primers guided by the synthesis of complementary DNA(complementary DNA, cDNA), and then using two primers as a template for cDNA in accordance with ordinary PCR methods, can amplify the sequence of the fully encoded gene without inclusions.
the efficiency of mRNAs copying into cDNA differently with the important parameters of reverse transcription PCR;
generally speaking, when engaging in an uneven mRMA group, the conditions used are to result in maximum final yield of cDNA synthesis, and the following parameters are important.
1. Reverse transcriptase has two different reverse transcriptases that can catalyze mRNA as a template, oligo (dT) as a primer, and synthesize cDNA strands complementary to mRNA.
a purified avian myelin celloblastoma virus (AMV), consisting of two peptide chains, polymerase activity and strong rna enzyme H activity, its most suitable temperature is 42 degrees C, the most suitable pH8.3.
can eliminate the blockage of reverse transcription in the secondary structure of mRNA at high reaction temperatures, however, the activity of high levels of RNA enzyme H inhibits both cDNA production and limits its length.
, the poultry-source reverse transcriptase formulation can be contaminated with nucleic acid endoenzymes that can cut DNA.
another type of virus derived from the mouse leukemia virus (Mo-MLV), which is monopeptide chain, has RNA polymerase activity and relatively weak RNA enzyme H activity, the most suitable temperature of 37 degrees C, the most suitable pH7.6, weaker RNA enzyme H activity to obtain 2-3kb mRNA full-length cDNA has great benefits.
the use of methyl hydroxide before the first chain reaction to destroy the secondary structure of mRNA, this step may be more important for the reaction of rat-derived reverse transcriptase catalysis at the lowest appropriate reaction temperature (37)C.
adding an excess of pyridoxatonic reagents before the first strand of synthetic cDNA can dissose zirconia from RNA.
2. The unit price cation ion condition basically affects the transcription efficiency of various templates.
can obtain longer transcription products with potassium than sodium ions.
the optimal potassium ion concentration for cDNA length is 140-150mM.
. Divalent cations are necessary for reverse transcriptase activity.
below 4mM Mg2 failed to observe activity;
4. The use of high concentrations in each of the four dnanucleonucleophosphates (dNTP) is particularly important for the effective synthesis of cDNA.
if only one of these concentrations drops below 10-50 micromoles, the yield of full-length transcriptions will decrease significantly. the concentration of dNTP commonly used in
is 200-250 micromoles.
experimental step 1. Material RNA samples.
2. Instruments, appliances PCR instrument, electrophoretic instrument, 0.2 ml PCR tube (1), pipette, broken ice.
3. Reagent RNase Free dH2O; 5 x RT Buffer (including 25mM Mg2) ;d NTP (10mM each); RNase E. (10U/?l); Oligo (dT) 20 (10 ?mol/L); ReverTra Ace.
4. Method (1) synthesizes the first chain of cDNA with the total RNA as a template.
(2) Reverse transcription reaction conditions are: 30 degrees C 10min, 42 oC 30min, 99 oC 5min, 4 oC 5min.
an ice bath for 5 minutes after the reaction.
5. Note cDNA first chain synthesis reaction liquid ice preparation.
use enzymes such as ReverTra Ace and RNase Inhibitor, should be gently mixed to avoid foaming, and because the enzyme preservation solution contains 50% of the glycerin, high viscosity, should be absorbed slowly when taking.
reverse transcription PCR, the extraction of RNA is the key, when collecting RNA precipitation, the centrifugal speed should not be less than 13000 rpm, pre-centrifugation is also as low as possible in low temperature conditions, in addition to the reverse transcriptase can choose MMLV reverse transcriptase, mainly because the condition is relatively easy to master.
Source: Algae Biodiscovery.