RNA extraction
Regarding RNA extraction, various academic groups and research institutions use different operational procedures, but the following factors are worth noting:
1. There are differences in RNA extraction methods for samples stored in different ways and in different forms;
2, the separation of total RNA, or the size of the 殌 RNA when the method of extraction is different;
3. Ensure that the RNA is extracted by centrifugation at 4 ° C. Because the temperature is not correct, it will lead to inappropriate stratification to produce the variant RNA product, and the whole extraction process needs to be completed quickly.
Reverse Transcription
The reverse transcription reaction can begin with random primers, oligo (dT) or gene-specific primers (GSP). The reverse transcriptase and RNase inhibitors used in the experiment will have a certain influence on the quality of the product, so the whole process requires no RNase operation.
Correct bifurcation of gene expression requires good reproducibility of reverse transcription. The reverse transcription reaction determines the sensitivity, kinetic range and specificity of the reverse transcriptase used. Reverse transcriptases derived from MMLV and AMV are the most widely used enzymes. For longer or full-length cDNA transcripts, MMLV reverse transcriptase and its derivatives are better than AMV reverse transcriptase because of their lower RNase H activity.
In two-step RT-qPCR, the reverse transcription step can use oligo (dT) primers, random primers, a combination of both or gene-specific primers. The choice of primers will affect your quantification of the gene of interest. The use of gene-specific primers has a lower background than using oligo (Dt) or random primers. If you choose oligo(dT) primers for reverse transcription, you should design the PCR primers near the 3' end of the transcript to avoid the decrease in sensitivity caused by mRNA shortening; this is especially important for longer transcripts.
qPCR amplification
Primer design
If the cDNA sample or genomic DNA sample is used as a template for amplification, it is suitable to design primers at 18~30bp; if the plasmid is used as a template for the sub-diplon, the primers are suitable between 35~40bp.
2. System selection
If the amplified fragment has no special junction, the general PCR amplification enzyme can be selected; if the amplified cassette has a high GC content and the content of the ruthenium in the sample is low, it is suitable for the high-GC content amplification enzyme effect. Better; high-assurance enzymes are more suitable for use in Aachen.
3. Reaction condition selection
If the amplified fragment has no special junction, it can be amplified by a three-step method; if the amplified fragment is high in GC content, a two-step method can be considered.
4. For the first amplification, it is best to use a gradient PCR to amplify at different annealing temperatures to select the most suitable temperature.
5. The length of the extension should be different. The details of the extension should be different. Please refer to the instructions of the selected system for details.
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