Rna 6000 nano labchip assay

Isolated germ-cell samples were resuspended in QIAzol Lysis Reagent according to the manufacturer's instructions, and total RNAs were purified from homogenized cells using Qiagen RNeasy Mini Kit (74104). The quality of the isolated RNA was assessed using an Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) and RNA 6000 Nano LabChip assay (5067-1511, Agilent Technologies).
The mRNA sequencing libraries were prepared using the Illumina TruSeq methodology. mRNAs were purified from total RNA using biotin tagged poly dT oligonucleotides and streptavidin coated magnetic beads. The mRNAs were then fragmented and double stranded cDNA was generated by random priming. The library was then analyzed for quality using an Agilent 2100 Bioanalyzer instrument (Agilent Technologies) and DNA 1000 LabChip assay.

Livers for RNAseq were harvested in the morning from chow-fed nonfasted B6 and HLB444 male mice that had been perfused with DEPC-treated PBS (N = 3 for each genotype). Tissue samples were stored in RNAlater (Thermo-Fisher) per manufacturer's instructions and homogenized in TRIzol (Invitrogen, Carlsbad, CA). Total RNA was isolated by TRIzol® Plus (Thermo-Fisher) according to manufacturer’s methods including on the column DNase digestion. The quality of the isolated RNA was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip assay. 500ng of total RNA was then reverse transcribed with random decamers and M-MLV RT using the Message Sensor RT Kit (Ambion, Austin, TX). mRNA was then purified from total RNA using biotin-tagged poly-dT oligonucleotides and streptavidin-coated magnetic beads followed by QC using the Agilent Technologies 2100 Bioanalyzer. The mRNA was then fragmented and double stranded cDNA generated by random priming. Illumina-specific adaptors were ligated to the fragments to create libraries, which were then sequenced on an Illumina HiSeq2000.

Total RNA was extracted from primary pituitary cells using the standard TRIzol (Invitrogen, Carlsbad, CA, USA) method according to the manufacturer’s protocol. Extracted RNA quality was assessed using the RNA 6000 Nano LabChip assay and an Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). Total RNA (1 μg) was transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and pd(N)6 random hexamer (GE Healthcare, Milan, Italy) in a final volume of 20 μl, according to the manufacturer’s protocol, as reported [31 (link)]. The primers used to verify the presence of Pit-1, FSHβ, LHβ, POMC, TSHβ, PRL, and GH in cultured pituitary cells were designed using mouse mRNA sequences, as previously described [29 (link), 32 (link)], and are listed in Table 1. The thermocycling conditions of PCR were: 95°C for 30s (denaturation), 60°C or 58°C depending on primer requirements for 30s (annealing), and 72°C for 1 min (extension).
All PCR-amplified products were visualized under ultraviolet light on a 2% agarose gel containing ethidium bromide. 1 kilobase (Kb) and 100 base pairs (bp) DNA Ladder (M-Medical, Milan, Italy) were used to determine the product size. All PCR fragments were sequenced to confirm identity.