Hello, I have been doing some transfection experiments for certain gene studies and wants to further do gene expression studies using qRT-PCR. Currently, I am using 7*10,000 cells per well for transfection and after 48 hours of transfection, I am isolating total RNA using kit method. But, my total RNA concentration is not good for further cDNA synthesis, as an example, I am getting concentration in the range of 10ng-250ng only. According to the cDNA synthesis protocol I need total RNA concentration at least 3-10ug.
How can I improve the total RNA concentration in order to achieve higher amounts for further cDNA synthesis? Your help will be very appreciable.
Hi Richa, are you using a cDNA synthesis kit? It is quite surprising that your cDNA synthesis protocol requires that much of RNA (3 to 10ug). I have been using different kits (QIAGEN and other brands as well) for cDNA synthesis step and it just needs RNA amount in the range of ng or even pg.
Kong Yen Liew Yes the Kit name is Takara Bio USA, Inc. Mir-X miRNA First-Strand Synthesis and TB Green qRT-PCR
Richa Pandey, I have no experience in using the kit that you mentioned. But since it needs that much of RNA, I think you have to scale up your assay by transfecting more cells (e.g. from 24-well to 6-well) in order to get more RNA. Or else you may want to opt for another kit that has a much higher efficiency (a kit that needs a lower input of RNA for cDNA synthesis).
Kong Yen Liew I wonder if my cell numbers are fine for transfection of I need to increase it ? Also, I am transfecting cells with the plasmid DNA having concentration range 150-300 ng, it can be the factor also for less total RNA concentration?
Richa Pandey, when you said your RNA concentration is in the range of 10 - 250 ng, are you meaning that it's 10 - 250 ng/uL?
If you increase your cell number definitely you have to increase the amount of plasmid DNA you use for transfection as well.
To me, the major portion of the RNA that you harvest would be from the cells rather than the plasmid DNA that you used, so I don't think that is the main reason you get low RNA concentration.
- Badges
- Science method