Abi prism 7900 ht detection system

For gene expression analysis, 10⁵ cells/well (ERMS) or 15 × 10⁴ cells/well (ARMS) were seeded in six-well-plates. After incubation of the cells for 24 h, total RNA was isolated using TRIzol Reagent (Invitrogen GmbH, Karlsruhe, Germany) and cDNA was synthesized using Superscript II and random hexamers (Invitrogen, Karlsruhe, Germany). Quantitative RT–PCR of target cDNAs was performed using SYBR-green based assays on an ABI Prism HT 7900 Detection System instrument and software (Applied Biosystems, Darmstadt, Germany). The primers for amplification of target transcripts are shown in the Table S2 in Supplementary Material. All primers used in study were intron-flanking, except of the primers for 18S and hMYOD. Expression levels of 18S rRNA served to normalize the transcript levels. Each sample was measured in triplicates. Expression of major components of the HH signaling pathway was analyzed once. All other data shown are the summary of two independent experiments performed in duplicates. Graphs represent the mean value of all measurements plus SEM.

RT-qPCR analysis was conducted as previously indicated [62 (link)]. In brief, total RNA was extracted from renal tissues or cells using Trizol reagent (Invitrogen, USA) following the manufacturer’s instructions. Then, the obtained total RNA was reverse transcribed using M-MLV-RT system (Promega, USA), which was performed at 42° C for 1 h and terminated through deactivation of the enzyme at 70° C for 10 min. Subsequently, PCR was conducted with SYBR Green (Bio-Rad, USA) on an ABI PRISM 7900HT detection system (Applied Biosystems, USA). All primer sequences used in the study were obtained from Invitrogen Corporation or Generay Biotech (Shanghai, China), and were listed in Supplementary Tables 1, 2. The quantification of each gene was analyzed according to the 2^-ΔΔCt expressions. ΔΔCt represents the relative change in the differences between the control and treatment groups. Expression of each mRNA was normalized with GAPDH mRNA.

The method measuring telomere length with real-time PCR was reported previously (Peng et al., 2011 (link)). Genomic DNA was extracted on the basis of the manufacturer's instrctions (Aidlab, Beijing, China). Quantitative RT-PCR was conducted using SYBR Green Reaction Mix (TaKaRa, Dalian, China) according to the manufacturer's instructions on an ABI PRISM 7900 HT Detection System (Applied Biosystems, Carlsbad, CA, USA). The primers were as follow (Peng et al., 2011 (link)): Telomere forward primer 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′, reverse primer 5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′. The reference gene 36B4: forward primer 5′-CTCACTCCATCATCAATGGATACAA-3′, reverse primer 5′-CAGCCAGTGGGAAGGTGTAGTCA-3′. PCR conditions were 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 56°C for 31 s (Peng et al., 2011 (link)). The single-copy gene 36B4 was used as a control and the target gene expression was calculated using the ΔΔCt comparative method.

Naïve CD8 T cells were purified from PBMCs using EasySep naïve CD8 T‐cell enrichment kit (Stem Cell Technology, Vancouver, B.C., Canada). Total RNA was extracted using RNeasy Plus Micro kit (Qiagen, Hilden, Germany) and reversed transcribed using Invitrogen Superscript VILO RT‐PCR Master Mix (Thermo Fisher Scientific). Real‐time quantitative PCR was performed with ABI Prism 7900HT Detection System (Applied Biosystems, Foster City, CA, USA) using Taqman Universal Master Mix II, no UNG (Thermo Fisher Scientific) and Taqman probe sets. Probe sets used are as follows: IL7R (Hs00902334_m1), CCR7 (Hs01013469_m1), SELL (Hs00174151_m1), TCF7 (Hs01556515_m1), and RPLP0 (Hs99999902_m1). All data are presented as relative expression normalized to RPLP0 expression, as this control gene is found to be expressed the most consistently between young and old naïve CD8 T cells.

Naïve CD4 and CD8 T cells were isolated fresh PBMCs using EasySep isolation kits (StemCell Technologies). RNA and cDNA was generated as described above. Gene expression was quantified using Taqman assays (Thermo Fisher Scientific) on an ABI Prism 7900HT Detection System (Applied Biosystems), and normalized to RPLP0 as a control gene. Taqman assays used in these studies are listed in Supplementary Table S3.

After counting, cells were suspended in a lysis buffer. Total RNA from harvested cells in different experiments was quantified using a ND-100 spectrophotometer (Nanodrop Technologies, Wilmington DE USA). RNA samples were reverse transcribed using the MMLV reverse transcriptase (Promega, Madison, WI USA) and oligo-dT primers. Real-time PCR amplification was done using 10 μL amplification mixtures containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA USA), equivalent to 4 ng of reverse-transcribed RNA and 150 nM primers. Reactions were run on an ABI PRISM 7900 HT detection system (Applied Biosystems) using a fluorescent threshold manually set to 0.150 for all runs. Table 1 shows the primers used for the estimation of gene expression; the Rpl32 gene was used as control of charge.
A semiquantitative approach for the estimation of the concentration of specific gene mRNAs per cell or unit of tissue weight was used [20 ]. The data were presented as the number of transcript copies per cell, allowing for direct comparisons independently of the number of cells in a given well.