cDNA was generated with the Takara RNA PCR Kit (AMV) with 500 ng of RNA as template and random 9-mers.
cDNA synthesis was carried out using a cDNA synthesis kit (Applied Biosystems Inc., Foster City, CA, USA) using 1 μg total RNA as the template and random primers.
We argue that this is not an annotation artefact, but most likely results from the end of template and reduced random priming potential in the first strand synthesis step amplification.
Positronium annihilation lifetime spectroscopy reveals that porous MSQ film templated by triblock copolymers (M̄n in the range of ∼5000 12,000 g/mol) have smallest pores and highest percolation threshold compared to those templated by diblock and random copolymers.
For all reactions, no-template controls were run, and random RNA preparations were also subjected to sham reverse transcription to check for the absence of genomic DNA amplification.
Total RNA was extracted from homogenized tissue according to the protocol provided in the RNeasy tissue Mini Kit (Qiagen, Hilden, Germany), reverse transcribed using 500 ng RNA as template and oligo-dT and Random Hexamers (Ration 1 2) as primers to obtain cDNA fragments.
The library construction was designed, and random oligonucleotide templates were synthesized as a single-stranded 88-mer with the following sequence: 5'-GCGGAATTCTAATACGACTCACTATAGGGAACAGTCCGAGCC-N30-GGGTCAATGCGTCATA-3', where the central N30 represents random oligonucleotides based on equal incorporation of A, G, C, and T at each position [37], [39].
RNA was purified and first strand cDNA was produced from 1 µg total RNA template using AMV reverse transcriptase and random primers (Promega) following the manufacturer's instructions.
For the SKI and chemokine templates, the random sequences are constrained to maintain the conserved, native cysteines.
