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=====Reverse Transcription===== *mRNA degradation --> I don't think this step has any problem. **I've cleaned bench and glass pipette for aspiration with RNAase-free and 70% ethanol, and UV the PBS, 0.1N HCl before starting. **After 0.1N HCl incubation for 10min, cells were washed with PBS 3 times. **'''To verify RT works well or not''', Cy5 labeled primer will be used to visualize cDNAs ***Primer for RT --> 5' 5Phos - TCT CGG GAA CGC TGA AGA NNN NNN 3' ***Labeled primer --> 5' Cy5 - TCT TCA GCG TTC CCT AGA 3' ***Order this primer, then hybridize after RT, and observe the signal. *Reaction time was 8 hour **Based on the polyA FISH protocol (1 hour hybridizatoin time) over 1 hour incubation time could ensure fully hybridization of primers to mRNA. **2000 units RTase in 200uL reaction mix are used, and 50 nmole dNTPs are used,--> 8 hr incubation time was very long time for cDNA extention. **Unit definition(from Enzymatrics --> 1 unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37Β°C using poly r(A)/oligo (dT) as a substrate. **'''To find better RT reaction time''', try different reaction time per each sample and use Cy5 labeled primer to see which sample has more cDNAs ***(First of all I need to verify that the way using Cy5 labeled primer works)
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