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RT- PCR Introduction
RT-PCR is a combination of RNA inverse transcription (RT) and c and ">DNA polymerase chain amplification (PCR). First, the role of reverse transcriptase synthesis from RNA cDNA and then cDNA as a template to amplification of the synthesis purpose fragments. RT-PCR technology is sensitive and versatile, and can be used to detect the level of expression ofgene in cells, the content of RNA viruses in cells and the cDNA sequence of specific genes cloned directly. RNA as a template can be a product of total RNA, mRNA, or in-body transcription of RNA.
No matter what RNA is used, the key is to ensure that there are no RNA enzymes andanddna contamination. Using Time's total RNA extraction systems (e.g. catalog numbers DP405 and DP406), the resulting RNA is highly pure, genomic DNA contamination is low, and satisfactory results can be obtained for RT-PCR systems.
select one of the randomized, Oligo dT, and gene-specific citations for the specifics of the visual experiment of the quotation used for reverse transcription. For short ethyrell mRNAs that do not have a card-issued structure, all three are available.
RT-PCR quotation selection
| random | .suitable for long or carded structures of RNA. Suitable for reverse transcription reactions of rRNA, mRNA, tRNA, etc. It is mainly used for RT-PCR reactions in a single template. |
| Oligo dT | applicable to tails with RNAPoly. (RNA from primary nuclear organisms, Oligo dT rRNA and tRNAs of ego organisms do not have PolyA tails.) Because Oligo dT is to be combined with PolyA's tail, the quality requirements for RNA samples are high, and even a small amount of degradation can significantly reduce the amount of full-length cDNA synthesis. |
| gene-specific quotations | < "td bgcolor" "#eefff0" height="54" style="text-align:left;"width"544"> and template sequence complementary quotations, suitable for the purpose sequence known. SuperScript One-Step System is particularly suitable for use with gene-specific citations.
RT-PCR reaction is affected by a number of factors, such as the concentration of magnesium sulfate, the temperature at which the quotation abodates, the number of amplified cycles, etc.
< p style is "text-align: left;" > recommends 0.5-3.0 mM (difference of 0.5 mM) of magnesium sulfate for preliminary experiments.is beneficial to the reaction when increasing the temperature of the adgression and extension for a quotation with a higher TM. Higher temperatures help reduce the binding of nonse specific pronitients, thus increasing the yield of specific products.
< p style is "text-align: left;" > target RNA can be observed by 40 rounds of PCR reactions. However, if the target RNA is too scarce, or if there are very few starting materials, it is necessary to increase the number of amplifications to 45-50 times.
