Total RNA (1 μg) was used for template of first-strand cDNA synthesis using extent reverse transcription kit (PrimeScript RT reagent Kit with gDNA Eraser, TaKaRa, Japan).
As the GeneChip system utilizes polyadenylation complementary oligonucleotides as a primer for reverse transcription of the starting RNA template, inefficiency of first strand cDNA synthesis and/or in vitro transcription of cRNA can result in under representation of the 5' moiety of the transcript [ 34, 52].
PCR reactions were performed as follows: one cycle at 94°C for 4 min; 25 cycles at 94°C for 30 s, 56°C for 40 s and 72°C for 30 s; followed by one cycle at 72°C for 10 min. The first round PCR products were used as the templates of second round inverse PCR after 100 fold dilution.
Romy Madley Croft, Oliver Sim and Jamie Smith may have stuck close to the template of their first album – Coexist is yet another barely there album in which the dawn seems far away – but it's hard to imagine anyone minding, so compelling is their signature sound.
Using the template of the first PCR reaction, allele-specific primer pairs was used to test for the presence of the allelic variants from FC27 and IC of the families of the msp2 region.
A 1-μl aliquot of the first-round PCR product was used as the template of the second-round PCR reaction.
The 140 fold diluted samples were used as a template for second step of PCR reactions.
Semiquantitative (competitive) PCR analysis was carried out using as template 10 ng of first strand cDNA primed with an oligonucleotide starting 97 bp downstream the ATG initiation codon of the iaaM gene, on mRNA extracted from flower buds.
RT-PCR analysis was carried out using as template 30 ng of first strand cDNA primed with an oligonucleotide starting 305 b downstream the AUG initiation codon of the iaaM gene, on mRNA extracted from flower buds.
One μl of DNA template in first PCR and one μl of first amplification products in second were screened, using the above mentioned primers and 0.25 μl of Taq DNA polymerase (NEB), following the manufacturer's instruction.
The Guardians Michael Cragg called "Trouble" a return to the "safer" templates of her first two albums, but praised the song's production calling it "a vaguely trip-hop beat buffeted by soaring strings and a troubled lyric in the chorus".
