Chapter 1 Introduction
2.6 RT-PCR and RT-qPCR
2.6.1 Poly-A tail-based reverse transcription and qPCR
For detecting the total pri-let-7 (the sum of pri-let-7A, pri-let-7B and
SL1-pri-let-7) by qPCR, up to 1 µg of total RNA was used and subjected into poly-A
tail-based reverse transcription. Briefly, the sample was mixed with reagents listed as
follows and heat at 65 °C then cool-down to 4 °C to anneal the universal RT-primer
containing a VN anchor, oligo(dT) and the universal reverse PCR primer sequence,
called URT primer, as previously described approach 95.
Annealing of poly-A tail-based URT primer
RNA sample (up to 1 µg) 10 µL
50 µM URT primer 1 µL
10 mM dNTP mix 1 µL
Nuclease free water 1 µL
Subsequently, following waiting at least 1 minute for incubation of the
reaction at 4 °C, reagents listed below were added into the tube and then the reaction
was subjected into reverse transcription.
Reagents for poly-A tail-based reverse transcription
Primer annealed RNA sample 13 µL
5X First-strand buffer 4 µL
0.1 M DTT 1 µL
Superase-In (Ambion, #AM2694) 0.5 µL
Superscript III (Invitrogen, #18080044) 0.5 µL
Nuclease free water 1 µL
Final volume 20 µL
The program for reverse transcription was initiated from activating the
enzyme by keeping the temperature at 25 °C for 5 minutes. Next, for best activity of
RTase, the temperature was set at 55 °C for one hour. Finally, to stop the reaction, heat
inactivation was performed by setting the temperature at 70 °C for 15 minutes. The
RNA templates were eliminated by adding 1 µL RNase mixture, which contains 2.5 unit
RNase H (NEB, #M0297) and 5 µg RNase A (QIAGEN, #19101), and incubating at 37
°C for 20 minutes.
To perform the quantitative-PCR, the KAPA SYBR FAST qPCR kit was used
according to the manufacture’s instructions. The reagents used in qPCR were listed in
the table below.
Reagents for poly-A tail-based quantitative-PCR
50X diluted cDNA 5 µL
Forward primer (10 µM) 0.4 µL
Reverse primer (10 µM) 0.4 µL
Nuclease free water 4.2 µL
Final volume 20 µL
For detecting the signals, the ABI Prism 7000 system was used and the
program was set as follows.
Program used for detecting poly-A tail based quantitative-PCR
Step 1 95 °C 3 minutes Activate enzyme
Step 2.1 95 °C 30 seconds Denaturing DNA
Step 2.2 60 °C 30 seconds DNA synthesis
Detect the signals at the end of this step at each cycle and repeat step 2 for 40 cycles
Step 3 60-95 °C Dissociation
The sequences of oligonucleotides used in this assay are listed in the table below.
URT primer
5’-AAC GAG ACG ACG ACA GAC TTT TTT TTT TTT TTT VN-3’
Total pri-let-7 forward (L1)
5’-GCA TCT ACC TCG ATT GGA CCT A-3’
eft-2 forward
5’-GTG CTA ATC CAC CTC TGG AA-3’
Universal reverse primer (R1)
5’-AAC GAG ACG ACG ACA GAC TTT-3’
2.6.2 Random priming-based reverse transcription and qPCR
This assay was used to examine the presence or abundance of mRNAs or
primary miRNAs. 10 µg of total RNA was treated with DNase I (Ambion, #AM1907),
in the final volume is 20 µL for at least 30 minutes at 37 °C. The DNase was inactivated
by adding 4 µL of inactivation reagent with constantly tapping of the tubes for 5
minutes at room temperature. Following brief centrifugation for a few seconds, 10 µL of
the supernatant was used for subsequent steps. The cDNA was synthesized by
SuperScript III reverse transcriptase (Invitrogen, #18080044) using random hexamers.
The detail information of reagents used in cDNA synthesis is listed in the table below.
Annealing of random priming-based primers
DNase treated RNA sample (around 5 µg) 10 µL
100 ng/µL Random hexamers 1 µL
10 mM dNTP mix 1 µL
Nuclease free water 1 µL
Following adding reagents, the tube was put into a PCR cycler and the
program was set as 65 °C for 5 minutes and cool-down to 4 °C at least 1 minutes to
allow the primers to properly attach the RNA molecules. Prior into the cDNA synthesis,
several reagents were added into the tube as listed below.
Reagents for random priming-based reverse transcription
Random hexamers annealed RNA sample 13 µL
5X First-strand buffer 4 µL
0.1 M DTT 1 µL
Superase-In (Ambion, #AM2694) 1 µL
Superscript III reverse transcriptase (Invitrogen, #18080044) 1 µL
The program for cDNA synthesis was initiated from 5 minutes at 25 °C to
activate RTase and then one hour at 50 °C for enzyme functioning and finally stopped
by inactivating the enzyme for 15 minutes at 70 °C. The RNA templates were
eliminated by adding 1 µL RNase mixture, which contains 2.5 unit RNase H (NEB,
#M0297) and 5 µg RNase A (QIAGEN, #19101), and following incubation at 37 °C for
20 minutes.
To perform the quantitative-PCR, the KAPA SYBR FAST qPCR kit was used
according to the manufacture’s instruction. The reagents used in qPCR were listed in
the table below.
Reagents for poly-A tail-based quantitative-PCR
10-50X diluted cDNA 5 µL
2X SYBR FAST master mix (KAPA, #KK4600) 10 µL
Forward primer (10 µM) 0.4 µL
Reverse primer (10 µM) 0.4 µL
Nuclease free water 4.2 µL
Final volume 20 µL
For detecting the signals, the ABI Prism 7000 system was used and the program was set
as follows.
Program used for detecting poly-A tail based quantitative-PCR
Step 1 95 °C 3 minutes Activate enzyme
Step 2.1 95 °C 30 seconds Denaturing DNA
Step 2.2 60 °C 30 seconds DNA synthesis
Detect the signals at the end of this step at each cycle and repeat step 2 for 40 cycles
Step 3 60-95 °C Dissociation
The sequences of oligonucleotides used in this assay are listed in the table below.
Primers used in random priming-based quantitative-PCR For C. elegans
SL1-pri-let-7_forward (L2) 5’-GGTTTAATTACCCAAGTTTGAGGC-3’
pri-let-7 and
SL1-pri-let-7_reverse (R2)
5’-CGCAGCTTCGAAGAGTTCTG-3’
pri-let-7_forward (L3) 5’-TCCTAGAACACATCTCCCTTTGA-3’
pri-miR-84_forward 5’-ATTTGGCGATGCGAGAAAGT-3’
pri-miR-84_reverse 5’-AGGCAGACGTATGATGAATA-3’
pri-lin-4_forward 5’-GACAATTTCTAGAGTTTTGGTTGG-3’
pri-lin-4_reverse 5’-CCTTTTCCCCGAATACCATT-3’
pri-miR-241_forward 5’-GTTCGGAATGGATTTTGGTTG-3’
pri-miR-241_reverse 5’-AGTGATGTTTCGATCTCCAC-3’
drsh-1_forward 5’-CGGACAAGACCGGAGAAGTA-3’
drsh-1_reverse 5’-CGTTTCCCAAACCTTTTTCA-3’
pash-1_forward 5’-GATTGCAGCGAATGATGAGA-3’
pash-1_reverse 5’-TCCTCAACCATTCCATCACA-3’
dcr-1_forward 5’-TGGTGGTGATGTCTCGAAAA-3’
dcr-1_reverse 5’-TCCCAACGTCAGCAAATGTA-3’
alg-1_forward 5’-TGCGCAGAAAGTATCGTGTC-3’
alg-1_reverse 5’-CTCTGGTGGCAGGTAGGTGT-3’
tbb-2_forward 5’-CAAATTCTGGGAGGTCATCTC-3’
tbb-2_reverse 5’-CATACTTTCCGTTGTTGGCT-3’
lin-42_forward 5’-TCTTGTTCACGTGCACCTTC-3’
lin-42_reverse 5’-GGCTCCGTCTGGCATAGTAA-3’
eft-2_forward 5’-TGTGTTTCCGGAGTGTGTGT-3’
eft-2_reverse 5’-CCATCGTCGTCTCCGTAAGT-3’
lin-41_forward 5’-GGATTGTTCGACACCAACG-3’
lin-41_reverse 5’-ACCATGATGTCAAACTGCTGTC-3’
gfp_forward 5’-ACCAGACAACCATTACCTGTCC-3’
gfp_reverse 5’-TCCCAGCAGCTGTTACAAACTC-3’
For mammalian cells
TRIM71_forward 5’-TGTGAGCTGCTGTGGAAGGT-3’
TRIM71_reverse 5’-GTCTTCAGCTCCTGCACCTG-3’
Renilla_forward 5’-GCCTAAGATGTTCATCGAGT-3’
Renilla_reverse 5’-TACTGCTCGTTCTTCAGCAC-3’