I am trying to perform gene expression analysis by qRT-PCR on very few (~1000) FACS sorted cells. The RNA yield is very less (1-5 ng/µl). This creates problems of very high cycle threshold (Ct) values (40
Dear Pooja, for low RNA yield samples, numerous strategies have been adopted by researchers based on their availability to increase RNA yield and thats what research means. So, you need to look for the options, which are easier to avail and can be adopted in your lab under your budget.
For less cell concentration, usually add less lysis buffer as compared to the recommended one in your protocol, during RNA extraction phase. If the protocol says 200 uL, use 100 to 150.
For low cells case, I personally recommend to do manual magnetic beads separation rather than column-based or Trizol-based methods. You will found a drastic improvement in terms of RNA yield achieved by magnetic beads.
After RNA extraction, often added is 0.1 M MgCl2 with prolonged incubation on ice for 1 hour to increase RNA yield and it is used especially for the Trizol method.
Addition of Glycogen (1 uL) along with Ammonium acetate and absolute ethanol after the extraction is recommended by many researchers. You can find the protocol easily.
Use of 4 M LiCl also increases RNA yield with some limitations. (Look for its protocol). Also, you can view my previous answers regarding the use of LiCl.
Important is to use maximum volume of RNA in uL for cDNA conversion, usually cDNA kits offers 9 - 10 uL of maximum RNA input. So utilize full conversion capacity of the kit.
Use the cDNA kit having SSIV enzyme with VILO mastermix (variable input linear output), which is best for fewer RNA transcripts samples, though, it is bit expensive.
In your qPCR, you can use maximum of 5 uL cDNA template, if encountering delayed Cts.
I hope these will help you out.
Regards....
Hi Pooja,
Nothing can work if RNA yield is too low. First, you need to increase RNA yield by reducing elute volume. For example, if the protocol recommends 50 ul, you need to reduce the volume to 10-15 ul. Second, I suggest one-step qRT-PCR using RNA as template instead of using cDNA, because reverse transcription from RNA to cDNA will lose much RNAs. Third, you can combine more cells from different sorting.
Hope it will work for you.
Thanks Han Zhang, I tried it.
Unfortunately, it did not make much of a difference. Since its a column-based elution, the lesser volume of 10-15µl may have made it more difficult to elute the bound RNA. And I got more or less a similar yield
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