Cfx connect real time system cycler

miRNA qRTPCR analysis was performed using Taqman assays (Applied Biosystems) (Cat #4429795): miR-145 (Assay #2278), miR-125a-5p (Assay #2198). The endogenous control was U6 snRNA (Assay #1973). Analysis of mRNA expression for Ret and DAT used (Cat #4331182, Rn00562224_m1) and (Cat #4331182, Rn01463098_m1) respectively. GAPDH (Cat #4331182, Rn01775763_m1) was selected as an endogenous control [56 (link)]. Analyses were performed with a final volume of 10 µL of miRNA cDNA, or 20 µL of mRNA cDNA and Universal PCR Mastermix (#4369016) in a Bio-Rad CFX Connect Real-time system cycler (Bio-Rad, CA, USA). Each sample was run in triplicate. Expression was normalized (∆Ct) using the appropriate endogenous control; small nuclear RNA U6 for the miRNA, and GAPDH for the mRNA analyses. A one-tailed T-test (comparison of means) was used to test for significance, in line with the differential expression observed for the array data.

RNA from intestinal tissue samples of 0 Gy and 5 Gy mice was extracted using the TRIZOL method. C-DNA was prepared with a c-DNA kit (iScript™ Select cDNA Synthesis Kit, Bio-Rad, Hercules, CA) as per the manufacturers protocol; Quantitative PCR was performed using specific oligonucleotide primers for NHE3, Lgr5, SGLT1, Erk, Akt Bmi1 and caspase-3. c-DNA (2 μL) was added to 18 μL of SyBr green mixture in CFX Connect Real-time System Cycler (Bio-Rad) for qPCR. One cycle consisted of 30 sec at 94 °C for denaturation, 60 sec for annealing, and 90 sec at 72 °C for extension. CFX Manager™ Software (Bio-Rad) was used for analysis. Standardization of the mRNA used the delta-delta Ct (DDCt) method. Briefly, DCt = Ct (target gene-treated) – Ct (ref gene-treated) and DCt = Ct (target gene-control) – Ct (ref gene – control). Therefore, DDCt = DCt (treated) –Ct (control). Fold change was calculated from the formula 2^(−DDCt).

For isolation of RNA 1 × 10⁶ spores were inoculated in 50 mL SXM and incubated for 3 d at 25 °C under constant agitation. RNA was purified using the Direct-zol RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. RNA integrity was tested by gel electrophoresis. Reverse transcription to cDNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Contaminations by gDNA were checked as described in [66 (link)] prior to quantification of gene expressions. Transcription levels were analyzed in triplicates (n = 1) using a CFX Connect Real Time System cycler (Biorad, Hercules, CA, USA) with Mesa Green qPCR MasterMix Plus for SYBR Assay (Eurogentec, Liège, Belgium). Primers JST325/326 (BIP1), JST290/JST291 (HAC1 variants), SZ9/SZ10 (H2A), and SZ11/SZ12 (EIF2B) were used. Expression levels of BIP1 and HAC1 were quantified relative to the reference genes histone H2A (VDAG_JR2_Chr4g01430a) and EIF2B (VDAG_JR2_Chr4g00410a) by qRT PCR using the ΔΔCT method [76 (link)]. Two independent transformants of HAC1ⁱ⁻HA and HAC1^u−HA were compared to a single HAC1 deletion, a HAC1-C transformant and wildtype in three independent experiments. Significances were calculated using two-tailed Student’s t-test.

Total RNA was isolated from cells of a DG-derived NPC line and from the spleen of 8-week-old female C57BL/6JRj mice using the RNeasy mini kit (Qiagen). 1 µg of total RNA was reverse transcribed with random hexamer primers using the Superscript II Reverse Transcriptase kit (Invitrogen). Quantitative real-time PCR was performed using the QuantiFast SYBR Green PCR kit (Qiagen). Each cDNA sample (100 ng/μl) was amplified using a Bio-Rad CFX Connect™ Real-Time System cycler, with primers specific for Xcr1 (Xcr1_fw 5′-GCACTGGAGGAGATCAAAGG-3′ and Xcr1_rev 5′-CGGGATGCAGGGATACTGAG-3′) or Itga9 (Itga9_fw 5′-ATGACGGGTTCCCAGATG-3′ and Itga9_rev 5′-TGTAGACTGCGCCAGCAA-3′). β-actin was used as an endogenous control (β-actin_fw 5′-TGACCCAGATCATGTTTGAGA-3′ and β-actin_rev 5′-GGAGAGCATAGCCCTCGTAG-3). Reactions with dH₂O in the absence of cDNA were included as a negative control for PCR amplification.