Abi prism 7900ht system

RNA was isolated using the High Pure RNA isolation kit (Roche, Mannheim, Germany). Reverse transcription was accomplished with the iScript Master Mix (BioRad, Munich, Germany). QRT-PCR was performed using SYBR green detection with mRNA-specific primers (Supplemental Table 1) on the ABI PRISM 7900HT system (Applied Biosystems, Darmstadt, Germany). Gene expression was analyzed with SDS, software version 2.4 (Applied Sciences) and normalized to the two housekeeping genes GNB2L1 and HPRT1.

Spinal cord samples from separate cohorts of mice were dissected and stored as described above. For real-time quantitative PCR, total RNA was isolated from spinal cord tissue using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The purity and concentration were determined spectrophotometrically. Reverse transcription was accomplished using a First Strand complementary DNA Synthesis Kit (Invitrogen). Real-time PCR was performed in an ABI prism 7900HT system (Applied Biosystems). All PCR experiments were performed using the SYBR Green I master kit (Applied Biosystems). All the primers were purchased from SABiosciences (SABiosciences). Quantification was accomplished according to the standard curve method. In order to achieve the same PCR efficiency for each analyte, 1:10 serial dilutions of cDNA were used to construct standard curves for Mpdz and GAPDH. The R² values for the standard curves for each analyte approached 1.0, suggesting the same amplification efficiency in the PCR reactions under these conditions. Melting curves were performed to document single product formation and agarose electrophoresis confirmed product size. As negative controls, RNA samples that were not reverse transcribed were run. Data were normalized to GAPDH mRNA expression.

Total RNA from empty vector (pcDNA) and Ror2-overexpressing (pRor2) cells was extracted using the High Pure RNA isolation kit (Roche). For each sample, 1 µg of RNA was transcribed into cDNA with the iscript cDNA synthesis kit (Bio-Rad). Gene expression was measured by SYBR green detection on the ABI PRISM 7900HT system (Applied Biosystems) from 10 ng cDNA per reaction with gene-specific primers. Data were analyzed with the SDS software version 2.4. (Applied Biosystems) and target gene expression quantified with the ΔΔct-method after normalization to the two housekeeping genes HPRT1 and GNB2L1. Primer sequences are as follows:
Ror2:

fw_5′-TTCTTCTTGGTTTGCATGTG-3′, rv_5′-CTGATCTCTTTGAGTTTGGC-3′

HPRT1:

fw_5′-TATGCTGAGGATTTGGAAAGG-3′, rv_5′-CATCTCCTTCATCACATCTCG-3′

GNB2L1:

fw_5′-AACCCTATCATCGTCTCCT-3′, rv_5′-CAATGTGGTTGGTCTTCAG-3′

LCP-1:

fw_5′-GCGGACATTTAGGAACTGGA-3′, rv_5′-GTATGGCGGTTTGTTTACTCTG-3′

FAT-1:

fw_5′-TCCTCCTGACATCATCTGCC-3′, rv_5′-GATAGATGCTCTCCTCAATTACCC-3′

VIL-1:

fw_5′-ATGAGCAGGAGAAGAAGGGA-3′, rv_5′-TCATTCTGCACCTCCACCT-3′

WIPF1:

fw_5′-GAAATGGCTTCCAAGACTCTC-3′, rv_5′-GTAGAATCTGCTTTCCCACTC-3′

HNF4G:

fw_5′-TATAGACTCCGTTCCCTACCA-3′, rv_5′-TTTCCTGTTGCTCTGTCCC-3′

This is an ongoing project for genetic study of NSOC, approved by the Ethics Review Committee (NJMUERC [2008] No. 20). The recruitment of study subjects was described preciously(Li et al. 2016). In brief, patients were recruited from three hospitals in Jiangsu Province between August 2008 and January 2015. Physical examination and medical records were obtained for clinical assessment. Patients with congenital isolated oral clefts without syndromic symptoms or other birth defects met the inclusion criteria of the case group. The controls were self-reported Chinese Han from the same region, and no known congenital anomalies were detected. Informed consent was provided by every volunteer of the study.
Approximately 2 ml of venous blood was collected from each participant and stored in a tube containing ethylenediaminetetraacetic acid. Genomic DNA was extracted by the phenol-chloroform method. With the ABI Prism 7900HT system (Applied Biosystems), all samples were genotyped by a TaqMan allelic discrimination method with a call rate of 98.6%. The sequences of the primers and probes are shown in Supplementary Table S1. Approximately 5% of the samples were randomly repeated, and the results were 100% concordant.

Following the applied treatment, the total cellular RNA of HUVECs was extracted by TRIzol, that was reversely transcribed using a ReverTra Ace qPCR RT kit (Toyobo). qPCR was performed under an ABI Prism 7900 HT System (Applied Biosystems, Foster City, CA). mRNA primers for Nrf2, HO1, NQO1, and GCLC were listed in Table 1. The product melting temperature was always calculated [40 (link)]. Quantization of the listed mRNAs was carried out through a 2^−∆∆Ct method, with GAPDH tested as the reference gene.

MBH samples derived from 14 different litters (n = 7 litters/maternal treatment) were harvested, immediately frozen in liquid nitrogen, and stored at −80°C. Tissues were homogenized and total mRNA was isolated using Trizol (Invitrogen). RNA products were reverse transcribed using reagents from Applied Biosystems. Quantitative PCR was conducted using Premix Ex Taq master mix (Takara) in an ABI Prism 7900 HT system (Applied Biosystems). Taqman Gene Expression assay FAM/TAMRA probes (Applied Biosystems) used for qPCR analysis were: Pomc (Mm00435874_m1); Cart (Mm00489086_m1); Agrp (Mm00475829_g1); Npy (Mm00445771_m1); Pcsk1 (Mm00479023_m1); Mcr3 (Mm00434876_s1); and Mcr4 (Mm00457483_s1). The expression level was normalized against the housekeeping gene Gapdh (Mm99999915_g1). Data were analyzed using the standard curve method.