RT-PCR for amplifying selected sequences
Total 10 µl of resuspended magnetic beads with bound mRNA-,
mRNA/cDNA-peptide conjugates were used as a substrate for 50 µl RT-PCR
reaction volume using PrimeScript One Step RT-PCR Kit Ver.2 with 0.4 µM
RTPCR-F and RTPCR-R primers (Supplementary Table 1). The reaction mix
was incubated at 80 ℃ for 30 seconds, then cooled down to 4 ℃ for primer
annealing, followed by the addition of PrimeScript enzyme and incubation
at 48 ℃ for 20 min to complete the reverse transcription reaction.
Reaction mix was then split into total 8 tubes to recover samples at
different PCR cycles (0, 5, 10, 15, 20, 25, 30 and 35) to check the
amplification efficiency. The PCR condition was set as follows: initial
denaturation at 94℃ for 60 second, followed by 30 cycles of 94℃ for 15
seconds, 62℃ for 30 seconds and 72℃ for 25 seconds, then final extension
at 68℃ for 30 seconds. In order to avoid over amplification of DNA, the
reaction samples were run on a 2% TAE agarose gel stained by SYBR Gold
and the optimal amplification cycle was determined based on the band
intensity and the absence of non-specific amplified products. Finally,
50 µl RT-PCR reaction was once again conducted with optimal PCR cycles
(20 cycles for round 1 and 2, 15 cycles for round 3) and the product was
purified using Wizard SV Gel and PCR Clean-Up System for the next round
of selection and sequencing.