I use U6 as internal control, and i found U6 sequence :
GUGCUCGCUU CGGCAGCACA UAUACUAAAA UUGGAACGAU ACAGAGAAGA UUAGCAUGGC CCCUGCGCAA GGAUGACACG CAAAUUCGUG AAGCGUUCCA UAUUUU
And i design the stem loop RT primer like this :GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA
and I use this three pairs of pimer to do the qPCR (SYBR GREEN )
U6 F1 CTCGCTTCGGCAGCACA
U6 R1 AACGCTTCACGAATTTGCGT
U6 F2:GCGCGT CGTGAAGCGTTC
U6 R2: GTGCAGGGTCCGAGGT
U6 F3: CGG GTGCTCGCTTCGGCAG
U6 R3: ATCCAGTGCAGGGTCCGAGG
But it could not be amplified.
The stem loop RT primer had been re-fold. And I have try different reverse transcriptase condition like 65℃ 10mins 42℃ 30mins or 16℃ 30mins 42℃30mins or 30℃ 10mins 42℃ 30mins. both of them do not work.
Could you please give me some suggestion? thank you