Q: At what preparation step should I bring over the samples?
A: Please bring over cDNA that has already been prepared in your lab. We recommend preparing at least 1 µg cDNA using qScript cDNA SuperMix (Quanta Bio Sciences).
Q: How much cDNA should I bring over?
A: This will vary depending on how many targets you wish to look at, but if you bring 5 µg this will insure that you should have enough.
Q: Do I need to OD my samples?
A: Yes. Original total RNA template should be quantified and the same amount of RNA should be used to make cDNA across your samples.
Q: Do I need to supply a positive control?
A: Yes, please provide us with a sample that is known to contain high levels of your target(s) so that we can use it to test the primers and to run a standard curve used for analysis.
Q: Do I need to DNase my samples?
A: Yes, the primers may pick up genomic DNA so to ensure you are reading only the RNA levels you must DNase treat the samples.
Q: Are there any good protocols to use for the sample preparation?
A: For preparing total RNA that does not include small RNA species, we recommend using Qiagen RNeasy kits. For small RNA isolation (such as microRNA) we have used Qiagen miRNeasy kits.
Q: Is it better to use Poly dT or random hexamers when making the cDNA?
A: The poly dT is slightly more efficient; however, if you plan to use 18s as a normalizer then you MUST use random hexamers.
Q: Do all my samples need to be prepared the same way?
A: Because of the sensitivity level of the real time and digital PCR, any differences in the preparation will be detected. All samples should be prepared at the same time and with the same protocol if you wish to compare them.