We have been assessing miR-155 in RNA recovered from FFPE DLBCL samples.
We found that using the TaqMan® standard Taqman miR kit, which require a separate RT reaction for every miR to be studied, we could detect this miR in RNA extracted from the FFPE samples, albeit that it amplified quite late, 30+ cycles. Similarly we could detect miR-155 in human MDM treated with M1 or M2 skewing agents, particularly in the former.
However, when we switched to the TaqMan® Advanced miRNA Assays, which allows for reverse transcription of all miRs in a sample at once, we were unable to detect miR-155 in RNA samples from any of our FFPE samples. This was specific to miR-155, as both miR-92a and miR-21 could be detected, when the qPCRs were run in paralell. Similarly, we could only detect miR-155 in M1 skewd MDM, at high cTs, but not at all in M0 and M2 MDM.
Does anyone have any ideas, could it be that low frequency miRs are missed by the bulk cDNA generation steps utilised in the TaqMan® Advanced miRNA kit?
Thanks in advance
Russell Foxall
hi,
Could you please let us know which master mix you have used for detection?
TaqMan® Advanced miRNA kit works with Taqman probes only. It does not work with SYBr green dye. I faced a similar issue, then I had to change the cDNA kit from Qiagen which works with SYBr green.
You can follow these discussions,
https://www.researchgate.net/post/Can_we_use_miRNA_cDNA_prepared_using_Taqman_advanced_miRNA_cDNA_synthesis_kit_with_SYBR_green_assay
Regards
Saurabh
Maybe the difference in the detection level is due to the "maturation"-state of miR-155? In the case of standard kit, you amplify a kind of pre-mature form of miR-155, whereas advanced kit amplifies already "processed" mature molecule, which level is lower?
You may be interested to read this:
https://www.researchgate.net/post/Can_we_use_miRNA_cDNA_prepared_using_Taqman_advanced_miRNA_cDNA_synthesis_kit_with_SYBR_green_assay
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