To check the expression of JAK2, STAT3, SRC and indoleamine 2 and 3 dioxygenase genes in the blood samples of people who do not have any immunological or hematological diseases, is the best way to isolate PBMC and check gene expression in PBMC or Can this be done in whole blood?
Hi Dr. Noormohammadi,
I think you are referring to mRNA as the same thing as RNA. A commonly used and reliable method for RNA extraction is the TRIzol. If you need further information, I would like to share the standard protocol with you. After RNA extraction, you can quantify the RNA concentration using a spectrophotometer and assess RNA quality with a bioanalyzer or gel electrophoresis. Once you have purified mRNA, you can use reverse transcription to convert it into complementary DNA (cDNA), followed by conventional PCR. The synthesized cDNA then can be used for the assessment of corresponding gene expression via real-time PCR.
Regarding the second part of the question, you can separate PBMCs by Ficoll density gradient.
Bests,
Dear Morvarid,
There are a number of methods to extract RNA. If you are intending to use an RNA extraction kit, any reliable company offers high quality kits. However, more conventional methods also result in high quality RNA. Depending on your aim (whether you test blood cells in an experiment or not) RNA can be extracted from blood and from isolated PBMC. In order to have high quality RNA, you must have highly viable cell pellet. Either extract RNA right after spinning your cells, OR snap-freeze using liquid nitrogen and store in a reliable -80 freezer until RNA extraction.
Good luck,
Regards,
ND
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning