Protoscript m mulv first strand cdna synthesis kit

E. coli MG1655 (WT) cells were transformed with plasmid pUH-C harboring one of the GFP-STM reporters. Cells were grown until reaching an OD₆₀₀ of 0.4 to 0.5 as described above and treated with NA (100 μg/ml) for 4 h. For RNA extraction, we used the RNeasy minikit from Qiagen as directed by the manufacturer. In order to discard DNA residues, we also used the RNase-free DNase set from Qiagen. From the RNA samples, cDNA samples were prepared by the use of the ProtoScript MMuLV first-strand cDNA synthesis kit (New England BioLabs). Synthesis was done by using the general primers provided with the kit. Next, in order to amplify the cDNA samples obtained, we performed PCRs using 3 different forward primers (PF) and one reverse primer (PR). PCR was set to only 15 cycles of annealing in order to be able to compare the quantities of the PCR products obtained.

For the induction of the lytic cycle, 2 × 10⁶ cells were stimulated with different concentrations of CPF for 4 h. The cells were washed three times with phosphate buffer saline (PBS) and then incubated in fresh culture medium for 48 h. The total RNA from the cells was extracted using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. The RNA was quantified through optical density measurement at 260 and 280 nm, and all of the samples had an A260/A280 ratio greater than 1.8. Equal quantities of the total RNA were reverse-transcribed into cDNA using a ProtoScript M-MuLV first-strand cDNA synthesis kit (NEB, USA). Real-time PCR was performed using a Maxima SYBR Green qPCR Master Mix (Fermentas, EU). The PCR primer sequences were obtained from qPrimerDepot (http://primerdepot.nci.nih.gov/) and synthesized by Shanghai Sangon (Shanghai, China). The data were analyzed through the comparative threshold cycle (Ct) method using GAPDH as the reference gene.

Strains were inoculated into YPAD and grown at 30°C for 16–18 hr. Cultures were then diluted 1:100 into YPAD and grown at 30°C for 4 hr. RNA was prepared using the MasterPure yeast RNA purification kit (Epicentre) according to the manufacturer’s instructions. RNA was treated with DNase (Epicentre) to remove contaminating genomic DNA. cDNA was prepared using the ProtoScript M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs) according to the manufacturer’s instructions with oligo dT primers. cDNA was measured by qPCR using the Universal Probe Library (Roche Applied Science) with a LightCycler 480 PCR machine (Roche Applied Science) or Rotor-Gene SYBR Green master mix (Qiagen) with a Rotor-Gene cycler (Qiagen) according to the manufacturer’s instructions. Expression was calculated as the amount of cDNA from the gene of interest relative to the amount of TEF1 cDNA in the same sample using the second-derivative maximum to determine C^T values and corrections for primer efficiency values.

Total heart or testis RNA purified using TRI reagent® (Sigma) was reverse transcribed into cDNA with Proto Script M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs, Italy), and the obtained cDNA served as a template for real-time PCR, based on the TaqMan methodology (Life Technologies). Primers (Applied Biosystems) and parameters are described in detail in Table 1. For evaluation of beta-galactosidase (β-gal) and FtMt transcripts, we used RT-PCR with the primers described in Table 1 and cycling conditions as follows: 5 min at 95 °C, 30 cycles (30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C), followed by 10 min at 72 °C.

Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen) and 1 μg total RNA was reverse-transcribed using the ProtoScript M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA). Quantitative PCR was performed using the SYBR Green PCR Master Mix on an ABI 7900 HT or an ABI StepOnePlus machine according to the manufacturer’s instructions (Invitrogen). Relative expression of each gene was determined using the ∆∆Ct method and normalized to the housekeeping gene Gapdh or Rps18 as indicated. Primer sequences used in this study are listed in Supplementary Table S1.

Samples for RNA isolation were harvested concurrent with samples for nChIP. Total RNA from HeLa Kyoto cells untreated, subjected to control siRNA (Luci) or PWWP2A siRNAs was isolated using the RNeasy Mini Kit (Qiagen) according the manufacturer’s instructions. One microgram of total RNA was reverse transcribed utilizing the NEB ProtoScript M-MuLV First Strand cDNA Synthesis Kit (NEB) according the manufacturer’s instructions. QPCR was performed on a LightCycler^® 480 Instrument II (Roche) using Fast SYBR Green Master Mix (Applied Biolabs). PCR efficiency and primer pair specificity was examined using a standard curve of serially diluted cDNA and melting curve, respectively. After normalization to the transcript level of HPRT1 (hypoxanthine phosphoribosyltransferase 1), data were analyzed based on the 2^−ΔΔCT method. For incorporating the standard error of the mean of the ΔΔCT values into the fold-difference, we calculated the range of the error bars as 2^{(–ΔΔCt + SEM)} and 2^{(–ΔΔCt - SEM)}. The following primer sets were used:
CCL5:
F: 5′ CTCGCTGTCATCCTCATTGC 3′
R: 5′ TACTCCTTGATGTGGGCACG 3′
FST:
F: 5′ TGCCTGCCACCTGAGAAAG 3′
R: 5′ TCTCCCAACCTTGAAATCCCA 3′
ZNF19:
F: 5′ CCCAGCAGAGAGGACCAAAA 3′
R: 5′ GCTGACCATGTGACATCATCC 3′
B3GALNT2:
F: 5′ TGGCTGCCATAGGACCTAA 3′
R: 5′ TCCACAGTTCCGTCAGTTCC 3′
CCDC71:
F: 5′ AAAGCTGCTGAAGTTCCGTG 3′
R: 5′ TGGAGCCGTATTACAGGTGA 3′