Hi Kelsie

you could be right: ct values > 30 are less than ideal but inevitable with low copy number genes

Howe consistent are your technical replicates for a given assay/sample ?

Hi,

I would suggest to not rely solely on the Ct values of the serial dilutions. The expression level of the gene may be low, but check also the 'melt-profiles' to see if the signal is due to your band or something undesirable and common component such primer dimers or so. Cross check by loading the samples on the gel also.

bye

Dear Kellsie

I will add 2 more things:

• To check that your samples perform well and consistently, try evaluating with a high copy number housekeeper; In fact I assume you might have already done this. If so how consistent are Ct values from your housekeepers ? If consistent that points towards something about detection of expression of your particular gene/isoform
• Is this variation a recent phenomenon or has this always been true ? If detectable expression has always been low  i.e. > 30 cycles for Ct, irrespective of how high or low the variance is within and between technical and biological replicates, check to make sure that your primers are pan specific, i.e. target an exon common to all identified Cyp genes: Many Cyp genes have multiple splice forms and these isoforms tend to be tissue specific so check to ensure that your are attempting to amplify all such isoforms by targeting a pan specific exon not a particular splice form that is expressed at low levels
• In addition, make sure you bias your primers to the last 2kb of your transcript; that is within 2kb of the Poly A site na dnot towards the 5' end of your elected transcript, especially if that ref Seq is > 3kb in length: When making cDNA reverse transcriptase proceeds 3' to 5' so the last 2kb of transcript are always amplified in to cDNA more efficiently than 5' regions
• This is especially true if you make cDNA with Oligo dT primers and not random hexamers: ALWAYS use randon hexamers for low levels genes but still situate your primers towards the 3' section of ther Ref Seq as degradation tends to proceed 5' to 3'. Have you checked the integrity of your RNA by Agilent biochip or on a denaturing agarose gel ?
• In addition, make sure your amplicon is ~100bp and no more than 200bp for low copy number genes: This will maximise efficient amplification  from low copy number genes
• Also add 5% DMSO to both your RT reactions and subsequent real time reactions; whether the latter are sybr green or Taq Man probes
• Finally, if all the above is being done and multiple primer sets have been tried then your issue is intrinsic low level expression and your only option might be to pre amplify from your cDNA before carrying out PCR or real time PCR: This is routine for low levels materials like Micr RNAs evaluated by real time PCR so many companies like Qiagen, Roche, Thermofisher etc. make reliable pre amplification kits

Hi Laurence,

Thank you for taking the time to respond to my answer. I have done qPCR with a reference gene and got tight for R^2 and a good range of Ct's. Nobody has examined the expression of this gene in this fish and I believe it is quite variable so I am not positive about the expression consistency. 

My transcript is only about 1.5 kb, however all of the primers I have been using are closer to the 5' end of the sequence so I may try ordering some closer to the 3' end and see what happens! The primers I have been using all produce a product under 150 bp so that should not be too much of an issue. Thank you for your advice!