Hey,
I am doing TruSeq RNA library Prep.
I extracted the RNA from Nasopharyngeal swabs (COVID-19) patients. The total RNA concentration is vary from sample to another. Also, the purity and degradation is vary and depends on sample transportation and handling from the hospital. However, when I reach the PCR step (last step) I follow the protocol and make 13 cycle of PCR. I ended up with low cDNA concentration. I raised to 15 cycles (15 recommended for bacteria) I get average 2ng/ul (some samples 0.5ng/ul and some about 10 or 20ng/ul.
What is the ideal number of PCR cycling for RNA extracted from Nasopharyngeal swabs (clinical samples)??
2ng/uL in what volume of buffer? This is very likely enough material to pool and sequence these libraries. I would suggest that you try eluting in a smaller buffer volume or concentrating the samples before doing more PCR.
Same happened to us. You will get very small amount of RNA. Better to use kit rather Trizol. Kits from Qiagen or MACHERY NANGEL are perfect and gave high yield. Do no cross > 20 cycles. It can insert mutations.
Hi Mohammed,
If you already isolated the RNA from all samples and cannot redo the isolation with another kit or lower the elution buffer like Laura and Waqar wrote, I would have another suggestion which, however, requires a slightly specialized PCR intrument to which you may or may no have the access. You could do the PCR step of library preparation in a real-time setup. For this, you would need a PCR machine which allows you to pause at any time, for example Biometra TOptical Gradient 96 or Qiagen Rotor Gene Q. If you then add a fluorescent dye to the PCR mix, e.g., SYBR Green or EvaGreen, you can actually monitor the PCR reaction in real time and end it when the PCR curve reaches a certain amount of fluorescence, for example 5000 FU, and take that sample out and leave the rest in the machine, resume the PCR and continue monitoring and taking samples out until the last sample reaches the desired fluorescence level. If there is another step of PCR after the cycling program, like final elongation, do not forget to finish this step with all samples. This way, you should be able to determine the optimal number of cycles for each library.
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