Hello Everybody,
I wanted to start this discussion to gather the experiment results from whoever have gone through this.
I am trying to extract total RNA from both serum and plasma, continue with cDNA synthesis and qPCR.
I am using grade II or III cancer patients’ blood sample.
I am on my validation period and still working on it.
Could you please share your experiments in this field ?
All you experiments such as:
- Serum and Plasma isolation protocol,
- Amount of serum and plasma used in RNA isolation,
- RNA extraction kit? Did you use specific kit for RNA isolation from serum/plasma or any other commercially available ones?
- Any key point in cDNA synthesis,
- Probe or syber green, which one did you chose for qPCR experiment?
- Any commercially available qPCR master mix or more specified one?
Thank you
Hi Samira Abdi
1. Which one (Serum or plasma) to prefer, that depends on your purpose. Serum can be prefered for Genomics & proteomics both while plasma majorly for proteomics. Isolation protocol will be available with the kit which you are going to use.
2. After isolation, how much RNA you get, based on that you can decide the RNA concentration to be converted into cDNA. Well, in a cDNA kit, the minimum and maximum amount of RNA needed or can be converted, those things will be given.
3. Yes, prefer a standard commercial kit for RNA isolation since samples are precious and need proper isolation. My suggestion look for Qiagen products.
4. cDNA synthesis will follow basic steps as per the manufacturer's instructions.
5. Which dye or probe, depending on your plan & budget. Both SyBr & Taqman work well as per standard principle.
6. TaKaRa, Qiagen, & Thermo all have qPCR master mix.
cDNA dilution, housekeeping genes expression, pipetting, qPCR experiment setup and qPCR data analysis, all these you will need to do properly.
Cheers!!!
In addition to what Saurabh Mandal suggested, it would be a good idea to check the document "Guidelines for Profiling Biofluid miRNAs" that can be downloaded here:
https://www.qiagen.com/-/media/project/qiagen/qiagen-home/clp/rna-resource-center/flyer/mirna_biofluids_guidelines_bro_0519_ww_1109075.pdf?cmpid=PC_QF_NON_rna-purification-traffic_0321_SEA_GA
https://www.hindawi.com/journals/jdr/2012/168368/
For extracting cfDNA you should utilize the assay with cfDNA extraction kits or viral DNA extraction.
For RNA assay you should add RIBOX or sth. like it, to prevent RNA from degradation.
qPCR is not an appropriate method .... you should quantitate it with the methods like HRM because of your goal (if you want to study the DNA methylation)
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