7900ht fast real time system

RNA was extracted from 10⁵ purified cells [TRI^® Reagent (Ambion)] followed by reverse transcription [High Capacity cDNA reverse transcription kit (Applied Biosystems)]. Bhlhe41 cDNA levels were quantitated by Taqman^® Gene Expression Assay predesigned primers (Mm00470512_m1) with intra-sample expression normalized to Eukaryotic 18S rRNA Endogenous control (FAM™/MGB probe) and run on an Applied Biosystems 7900HT Fast-Real Time System using SDS software (v2.4). Data were analyzed using the comparative CT method (Δ/ΔCT), where fold differences in gene expression between FOB and other B cell populations (ΔCT) were normalized to CT values of the 18S rRNA reference gene.

Total mRNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was converted to cDNA using a reverse transcriptase kit (Promega, Madison, WI, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences are listed in Supplementary Table S2. Quantitative polymerase chain reaction (qPCR) was carried out in triplicate in 96-well plates using a 7900HT Fast Real-Time system (Applied Biosystems, Foster City, CA, USA). The comparable cycle threshold method (2^-⊿⊿CT) was used to calculate the relative expression levels of the target genes.

Trizol reagent (Invitrogen) was used to extract total RNA from the tissues or cells. First-strand cDNA was synthesized using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Shiga, Japan). Real-time PCR was performed according to the protocol of the SYBR Green PCR Mix (Takara) using the 7900HT Fast Real-Time System (Applied Biosystems, Forster City, CA, USA). The sequences of the primers were: for Ednrb(GenBank: L06623.1): sence, 5′-GGGCTGCAGGTTTCGACC-3′ and antisence, 5′-CTGCAAACGCTAATACCGCC-3′; for Kng1(GenBank: BC060039.1): sence, 5′-CCTTTGGAATGGTGATACCG-3′ and antisence, 5′-CGCAAATCTTGGTAGGTGGT-3′; for caspase1(GenBank: NM_001257118.2): sence, 5′-TTTCCGCAAGGTTCGATTTTCA-3′ and antisence, 5′-GGCATCTGCGCTCTACCATC-3′; for GAPDH(GenBank: NM_002046.6): sence, 5′- CGGAGTCAACGGATTTGGTCGTAT-3′ and antisence, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. The PCR conditions were as follows: 95°C for 3 min; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles. GAPDH was used as internal control and relative gene expression was calculated using 2^−ΔΔCt (Livak and Schmittgen, 2001 (link)).

RNA was extracted using Qiagen RNeasy mini kit (Qiagen) and 500 ng/sample was used for the generation of cDNA using High Capacity cDNA Reverse Transcription Kit (Life Technologies) according to manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed on a 7900 HT Fast Real-Time system using TaqMan Gene expression assays (PPARC1A: Hs01016719_m1, TFAM: Hs01082775_m1, ESR1: Hs00174860_m1) and TaqMan Fast Universal PCR Master Mix (all from Applied Biosystems/Life Technologies) with the following cycling conditions: 95°C for 20 sec, 40 cycles of 95°C for 1 sec and 60°C for 20 sec. Expression of ACTB (Hs99999903_m1) was used as an endogenous control and the fold gene expression was calculated using the 2^−ΔΔCT-method [28] (link). Raw data are shown in file Raw Data S1.

TaqMan Rodent MicroRNA Arrays 2.0 (Life Technologies) were utilized for qPCR based miRNA expression profiling. We performed three separate reverse transcription reactions per sample using 150 ng total RNA each and Megaplex RT Primers Rodent Pool A and Rodent Pool B (Life Technologies) following the manufacturer’s protocol. Subsequently, each reverse transcription reaction was pre-amplified using the Megaplex PreAmp Primers Rodent Pool A and Rodent Pool B (Life Technologies) according to the manufacturer’s instructions. For each biological sample the three separate reverse transcription and preamplification reactions were pooled. The qPCR reaction mix was prepared according to the manufacturer’s instructions. The arrays were run on an Applied Biosystems 7900HT Fast Real-Time System with cycling conditions according to the manufacturer’s protocol. SDS 2.3 software (Life Technologies) was applied for obtaining raw expression data. The SDS files were loaded into the RQ Manager 1.2 software (Applied Biosystems, Life Technologies). Each amplification plot was reviewed manually. The threshold was set to 0.2 and adjusted for individual assays if necessary. We retained miRNAs which showed Cq-values smaller than 32 in at least two of the corresponding triplicates of a group for further data processing. We applied loessM normalization [22 ,23 ] using R programming [24 ].

Total RNA was extracted from INS1 832/13 and EndoC-βH1 cells using RNAeasy (Qiagen, Hilden, Germany) before complementary DNA (cDNA) was synthesized using SuperScript (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Concentration and purity of total RNA were measured with a NanoDrop ND-1000 spectrophotometer (A260/A280 > 1.9 and A260/A23 0 > 1.4) (NanoDrop Technologies LLC, Wilmington, DE, USA). RNA Quality Indicator (RQI) higher than 8.0 (Experion Automated Electrophoresis, Bio-Rad, USA) was considered to be high-quality total RNA preparation. TaqMan mastermix from Applied Biosystems (Foster City, CA, USA) was used for qPCR and performed following manufacturer’s protocol and was run in a 7900 HT Fast Real-Time System (Applied Biosystems). The qPCR was carried out as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Changes in gene expression were calculated using the ΔΔCt method with a fold-change cut-off at ≥ 1.5 and p < 0.05 considered significant. All samples were run in duplicate, and relevant negative controls were run on each plate. qPCR results were normalized to housekeeping genes (PPIA or HPRT). Primer sequences used in the qPCR assays are provided in Supplementary Table S2.