Abi 7500 rt pcr machine

The EM kit was prepared as described in the instructions for use (IFU)⁴⁰ . Briefly, dilution buffer was added to the lyophilised mastermix and vacuum-dried positive control. Resuspended mastermix was then dispensed into 48 wells/96 well plate. Eluate (10 µL), positive control, or dilution buffer (no template control) was added to the mastermix (final 20 µL reaction volume) and transferred to the RT-PCR machine (Applied Biosystems ABI 7500 RT-PCR machine or BioRad CFX96 Opus RT-PCR machine). Thermal cycling was performed for 10 min at 45 °C (RT step), 3 min at 95 °C (polymerase activation step), with 45 cycles 15 s at 95 °C (denaturation) and 30 s at 60 °C (annealing and polymerisation). Data were analysed for FAM (N1), JOE/HEX (RdRp), and ROX (Human RNaseP), with detection of either N or RdRp gene within 40 cycles (Ct) used for a positive call. For negative samples, detection of RNaseP within 40 Ct was required, otherwise the sample was determined as invalid. Positive (SARS-CoV-2 gRNA or supplied positive control) and negative (dilution buffer) amplification controls were performed with each RT-PCR amplification.

Total RNA was isolated from the liver samples of NCD group, GDM group and Inulin-H group using Qiagen RNeasy® Mini Kit (QIAGEN, Shanghai, China) according to the manufacturer’s instructions. Single-strand cDNA was synthesized from 1 µg of total RNA by reverse transcription reaction using Qiagen RT² First Strand Kit (QIAGEN, Shanghai, China). The cDNA was mixed with Qiagen PCR RT² SYBR Green Master Mix (QIAGEN, Shanghai, China).
To explore the underlying mechanisms of inulin induced-effects, the expression of 84 genes involved in Glycolipid metabolism including RETN were examined using RT² profiler PCR array (PAMM-006Z-mouse glucose metabolism, QIAGEN, Shanghai, China). Relative quantification of mRNA levels was determined by real-time quantitative PCR using a ABI 7500 RT-PCR machine/Bio-Rad CFX96 Sequence Detector instrument. The quantitative expression of gene was calculated from the cycle threshold (CT) value of each sample in the linear part of the curve using the relative quantification method (2^−ΔΔCT) [27 (link)]. The samples were analyzed in triplicate and corrected for the selected internal standard which had the smallest standard deviation among the housekeeping genes. Candidate genes were selected from those whose expressions differed greater than twofold or less than twofold, or which differed significantly (p < 0.05) between the GDM group and Inulin-H treatment group.

Total RNA was extracted using TRIzol extraction reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. RNA purity and concentration were photometrically tested. RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Takara, Japan). qPCR was performed using SYBR Green qPCR Supermix (Takara, Japan) with an ABI 7500 RT–PCR machine (Bio-Rad, CA, USA). The gene expression levels were calculated relative to β-actin using the 2^−ΔΔCq method. The cycling conditions were as follows: incubation at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 60 s, and 95°C for 15 s. The primer sequences were as follows:
β-actin F: 5' ACATCCGTAAAGACCTCTATGCC 3', R: 5' TACTCCTGCTTGCTGATCCAC 3'; Pdyn F: 5' CACGGAACTGACCAAGCTCT 3', R: 5' GTCAGTGCCCAGTAGCTCAG 3'; Mapk1 F: 5' TGAAGACACAGCACCTCAGCAATG 3', R: 5' GGTGTTCAGCAGGAGGTTGGAAG 3'; Pcdh9 F: 5'GTGCTTGGTTTTGGGTCACT 3', R: 5' CGGTCATTGAACTGGTTCCT 3'; Gnai1 F: 5' GTGCTTGGAGCCCGCACTCGG 3', R: 5' AGATTCACCAGCACCGAGCAGCA 3'; Pi4bk F: 5'GCCCACCAGGGAATAA3′', R: 5' TCCACTACTGTATCTCCCAT3'; Mhl3 F: 5' GACGTATGTTCCCGATTTTGTCA 3', R: 5' GCTTCAGAGCTGATATAGCCACT 3'; Ppp1cb F: 5' TGGACAGCCTCATCAC 3', R: 5' TTCAGCTCCCCGTCCGCCAT 3'.