Absolutely rna mirna kit

The cell bodies of CGCs were identified and individually dissected from the CNS of Lymnaea as described previously^{9 (link)}. Total RNA extracted from the CGCs (n = 10) by means of the Absolutely RNA miRNA kit (Agilent Technologies) was split into separate halves to analyse the expression of Lym-miR-137 and Lym-CREB2 mRNA.

The frozen (− 80 °C) cerebral and the buccal ganglia dissected from experimental (paired) and control snails (unpaired and naïve) (n = 20 in each group) were used to extract RNA by means of the Absolutely RNA miRNA kit (Agilent Technologies). The isolated RNA samples were treated with DNase TURBO (Ambion) to remove any traces of DNA and then quantified using the NanoDrop microvolume spectrophotometer. RNA integrity was confirmed by gel electrophoresis. The purified RNAs were copied into cDNAs using the iScript cDNA synthesis kit according to the manufacturer’s protocol (Bio-Rad). cDNAs were amplified and analyzed on the Mx3000P real-time cycler (Stratagene) using the Biotool SYBR Green qPCR Master mix (Stratech). Cycling parameters for the PCR were as follows: denaturation, 95 °C, 15 s; annealing, 52 °C, 20 s; extension, 60 °C, 20 s. We used primers 5′- GTAATAAGCGCATTTGCATAC-3′ and 5′- CCTGGTGTGAAGCTGATC-3′ for detection of Lym-NOS1AS (accession number MW300420, the amplicon size is 108 bp), and primers 5′-AAGGGACATTACACAGAGG-3′ and 5′-GTGTCAGTTGGAATCCTTG-3′ for detection of β-tubulin. The amount of Lym-NOS1AS NAT, normalized to an endogenous reference (β-tubulin mRNA) and relative to a calibrator, was calculated as 2^−ΔΔCT.

Total RNA was extracted from BMP4- and vehicle-treated cells using the Absolutely RNA miRNA kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. RNA quality was monitored using Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing libraries were generated using the TruSeq RNA Library Prep kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s directions. Shortly, total RNA was enriched for Poly-A tails and then fragmented. Subsequently, RNA fragments were reverse transcribed into cDNA using random hexamer primers. Then, short fragments were purified and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were ligated to sequencing adapters. Finally, suitable fragments were selected for the PCR amplification as templates and separated with agarose gel electrophoresis before sequencing.