Rneasy total rna isolation kit

mRNA expression in whole testes from P14 CT or Cfp1^Stra8 mice (n = 3/group) was compared using an RNeasy total RNA isolation kit (Qiagen) according to the manufacturer’s instructions. Biotinylated cRNA samples were prepared using 500 ng of total RNA according to the standard Affymetrix protocol (Affymetrix, Santa Clara, CA, USA). After fragmentation, 15 μg of RNA was hybridized at 45 °C for 16 h on a GeneChip Mouse Genome 430 2.0 Arrays. GeneChips were scanned using an Affymetrix GeneChip Scanner 3000 7G. The data were analyzed with RMA using Affymetrix default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized and log-transformed intensity values were then analyzed using GeneSpring GX 12.5 (Agilent Technologies, Santa Clara, CA, USA). Hierarchical clustering data were used to cluster groups that behaved similarly across experiments using GeneSpring GX 12.5 (Agilent Technologies). The clustering algorithm was Euclidean distance and average linkage. The statistical significance of differentially expressed genes (DEGs) was determined when the difference in gene expression between Cfp1^Stra8 and wild type had a p value ≤ 0.05.

H1N1pdm09 (200 μl) containing cell culture supernatant (1 × 10⁶ FFU/ml) was incubated with 10⁵ U/ml binase (final concentration) for 30 min at 37 °C. As a control, virus was incubated without binase. The viral RNA was then extracted and purified using “RNeasy total RNA isolation kit” (Qiagen, Germany) according to the manufacturer’s instructions. Then, vRNA of PB2 was amplified using “SuperScript III One-Step RT-PCR Platinum Taq High Fidelity” (Invitrogen, USA). Briefly, 100 ng of extracted vRNA were mixed with 25 μl “2× Reaction Mix”, 2 μl (0.4 μM) of forward and reverse universal primers [29 (link)]. The total volume was adjusted to 50 μl using nuclease-free water and then subjected to cDNA synthesis at 55 °C for 40 min, followed by pre-denaturation (94 °C for 2 min), PCR amplification (20, 25, 30, 35 cycles: 94 °C/15 s for denaturation, 58 °C/30 s for annealing and 68 °C/3 min for extension) and final extension (1 cycle: 68 °C/5 min). The RT-PCR products were detected by 1% agarose gel electrophoreses and gel documentation.

Total RNA was isolated from tissues or cells using TRIzol Reagent (Invitrogen, Shanghai, China) and purified using the RNeasy Total RNA Isolation Kit (Qiagen, Hilden, Germany). Real-time quantitative PCR was performed using the Real-Time PCR System (Biosystems, CA, USA) and the Perfect Real Time Kit (SYBR, Dalian, China). For the rat gene expression, the following SYBR Green real-time PCR primers were used: ZO-1 forward, 5′-AGTTCTGCCCTCAGCTACCA-3′ and reverse, 5′-GCTTAAAGCTGGCAGTGTC-3′; and β-actin forward, 5-CCTAGACTTCGAGCAAGAGA-3′ and reverse 5′-AGAGGTCTTTACGGATGTCA-3′.

Total RNA was extracted from samples with an RNeasy Total RNA Isolation Kit (Qiagen, 74104). Then total RNA was reverse transcribed into cDNA using a SuperScript^TM III First-Strand Synthesis System (Invitrogen Thermo Fisher Scientific, 18080051) according to the manufacturer’s instructions. The primers used are the same as previously published [12 (link), 33 (link)].

Real-time PCR was conducted as described previously 38 . Total RNA of cells or heart tissues was harvested using a TRIzol Reagent (Thermo Fisher Scientific, Waltham, USA) and a RNeasy Total RNA Isolation Kit (Qiagen, Hilden, Germany). Isolated RNA was reverse-transcribed using an iScript cDNA Synthesis Kit (Takara BIO, Otsu, Japan). Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) in the CFX96TM Real-Time System (Bio-Rad Laboratories, Hercules, CA). All primers were purchased from Sangon Biotech Corporation (Shanghai, China) and were presented in Table S2. Levels of miR-1 were measured using the mirVana qRT-PCR miRNA Detection Kit (Thermo Fisher Scientific, Waltham, USA) in conjunction with real-time PCR. The mimics and antagomir of miR-1 were both purchased from Thermo Fisher Scientific (Waltham, USA). Mutated nucleotides in the TBC1D15-3'UTR were conducted according to a previous report 39 . Luciferase activities were detected using a dual luciferase reporter assay kit (Promega) with a luminometer after 1 g PGL3-target DNA and 0.1 g PRL-TK transfection with lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA) for 48 h 40 (link).

Total cellular RNA was extracted from cells using RNeasy total RNA isolation kit (Qiagen; Hilden, Germany) following the manufacturer’s instructions, and then quantified using a NanoDrop™ One (ThermoFisher Scientific).
For the RNA viral gene NS5B detection, reverse transcription (RT) and PCR were combined in a single step as previously described [24 (link)].
Primary PCR was performed using the following specific primer pairs:
SENSE A: 5′-AAGATCCACCCTTATGA(A/G)GC-3′
ANTISENSE A: 5′-AAGAAGCCATCATC(A/C)CCACA-3′
The product of the primary PCR was used in nested PCR. The multiplex primers used for nested PCR are the following:
BVDV-1: 5′-TGGAGATCTTTCACACAATAGC-3′
MULTISENSE: 5′-GCTGTTTCACCCAGTT(A/G)TACAT-3′
For internal sample quality control, a volume of 1 μg of RNA was reverse-transcribed, in accordance with the manufacturer’s protocol. Qualitative PCR were performed using the following specific primer pairs for GAPDH:
F: 5′-CAACGGATTTGGTCGTATTG-3′
R: 5′-GGAAGATGGTGATGGGATTT-3′
Products were electrophoresed on a 1.5% agarose gel (ThermoFisher Scientific) and stained with gel red (Biotium, Hayward, USA).