The complementary strand of DNA (cDNA) was synthesized using oligo (dT)25 (ThermoFisher Scientific, Waltham, MA, USA). Total RNA extracted was normalized to 200 ng/μl and mixed with 6.4 pmol/μl oligo (dT)25 and 1 mM dNTPs (ThermoFisher Scientific), then incubated at 65 °C for 5 min. Then, 0.5× RT buffer (ThermoFisher Scientific), 1 U/μl RNase inhibitor (ThermoFisher Scientific), and 10 U/μl RT NxGen™ M-MuLV (Lucigen, Middleton, WI, USA) were added to the mixture and topped up with nuclease-free water to a total volume of 20 μl. Polymerase chain reaction (PCR) conditions were set at 42 °C for 50 min and then at 85 °C for 5 min using a GeneAmp® PCR System 9700 thermocycler (ThermoFisher Scientific).
Rt buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
RT buffer is a solution used in the preparation of samples for reverse transcription (RT) reactions. It serves as a buffer to maintain the appropriate pH and ionic conditions necessary for the reverse transcriptase enzyme to efficiently convert RNA into complementary DNA (cDNA).
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (12 mentions)
Rnaseout, by Thermo Fisher Scientific (9 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (9 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Dntps, by Thermo Fisher Scientific (7 mentions)
Rtase, by Thermo Fisher Scientific (7 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (6 mentions)
Random hexamer, by Thermo Fisher Scientific (6 mentions)
Rneasy micro kit, by Qiagen (6 mentions)
Rnaiso plus, by Takara Bio (6 mentions)
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Rnase free depc, by Takara Bio (5 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (4 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Random primer, by Thermo Fisher Scientific (4 mentions)
Lightcycler 480 real time pcr system, by Roche (4 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (4 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (4 mentions)
Oligo dt primer, by Thermo Fisher Scientific (3 mentions)
Rnasin, by Promega (3 mentions)
58 protocols using rt buffer
1
Reverse Transcription of Total RNA
2
Viral RNA Extraction and RT-PCR for Dengue Detection
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Viral RNA was extracted from 250 µl of whole blood using Trizol LS reagent (Thermo Fisher Scientific Inc., USA) following the manufacturer’s instructions. Extracted RNA was converted into complementary DNA (cDNA) using the SuperScript IV Reverse Transcriptase enzyme (Thermo Fisher Scientific Inc., USA). Reverse transcription-polymerase chain reaction (RT-PCR) was done in a 20 µl reaction mixture containing 10 µl extracted RNA, 0.1 µM reverse primer SBD2 [27 (link)], 1X RT buffer (Thermo Fisher Scientific Inc., USA), 10 mM dNTP mix (Sigma-Aldrich, USA), RNase Inhibitor (Thermo Fisher Scientific Inc., USA), and Hi-grade RNase-free water (GCC-Biotech, India). The RT-PCR cycling program involved single cycle heating at 90 °C for 10 min, reverse transcription reaction at 42 °C for 60 min, and enzyme inactivation at 72 °C for 15 min. The prepared cDNA was stored at − 20 °C for Dengue detection and serotype identification by polymerase chain reaction (PCR).
3
Analyzing NIPBL and WAPL Expression in HCT116 Cells
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HCT116 derived RNA was isolated using the RNeasy Plus Kit (Qiagen) according to manufacturer’s instructions. For complementary DNA (cDNA) synthesis, a 50μl reaction containing 20μl RNA, 1500pmol Oligo dT primer (IDT), 1.6mM dNTPs, 1x RT Buffer (Thermo Scientific), 0.5μl RNase OUT (Invitrogen), and 0.7μl Maxima RT (Thermo Scientific) was incubated at 50°C for two hours then at 85°C for 5 min. Samples were stored at -20°C until use. RT-PCR was performed using PowerUP Sybr (ThermoFisher, #A25741) based on manufacturer’s instructions. Briefly, cDNA was diluted to a working concentration of 6μg and HCT116 genomic DNA (gDNA) was diluted in a 1:10 serial dilution. A 6μl reaction was prepared per well, with 1x PowerUP Sybr and 0.2μM of the forward and reverse primers and combined with 4μl diluted DNA. Each reaction was performed in triplicate. qPCR was performed on the QuantStudio7 Flex System. YWHAZ and TBP were used as reference control genes. The sequences of oligonucleotides used for qPCR are: NIPBL forward primer: 5’-TCTCTTTGTTACTTGTCTGTTTCC-3’ and reverse primer 5’-ATGTTTTGCTTTGAAAACCAGTG-3’; WAPL forward primer 5’-GAACTAAAACAGCTCCATCACC-3’ and reverse primer 5’-CACACTTTCAGGCACACCAG-3’; YWHAZ forward primer 5’-CCCGTTTCCGAGCCATAAAAG-3’ and reverse primer 5’-TTTGGCCTTCTGAACCAGCTC-3’; and TBP forward primer 5’-ACAGCTCTTCCACTCACAGAC-3’ and reverse primer 5’-ATGGGGGAGGGATACAGTGG-3’.
4
Quantitative Analysis of Gene Expression
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Cell samples were lysed in 1:1 (v/v) RLT buffer (Qiagen) and 96% ethanol. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was converted into cDNA using Oligo d(T) 18 mRNA Primer (New England BioLabs), dNTP Mix, RiboLock RNAse Inhibitor, RT Buffer, and Maxima Reverse Transcriptase (all from Thermo Scientific). For real-time quantitative PCR (RT-qPCR), 30 ng of cDNA was used with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The primers were hTSPO (Bio-Rad, sequence not available), hTBP [31 ], and hRPLP0 [32 (link)]. Data analysis was done from Ct values normalized to the average value of two housekeeping genes (TBP and RPLP0). Results are shown as fold-change expression to Ctrl (delta-delta Ct method).
5
Comprehensive gene expression analysis
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The reagents benzaldehyde, diacetyl, methyl-salicylate, Nile Red, anthranilic acid, and paraquat-dichloride hydrate were obtained from Sigma Aldrich. For quantitative gene expression analysis, primers were purchased from Sigma Aldrich. The GeneJet RNA purification kit, Proteinase K, Maxima RT enzyme, Ribolock RNase inhibitor, RT buffer, and dNTP were acquired from Thermo Scientific. Random Hexamer was from Invitrogen.
6
RT-qPCR Gene Expression Analysis
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Four microliter of the reaction‐containing DNA‐free RNA were mixed with 0.5 μg (1 μl) of oligo(dT)18 (ThermoFisher Scientific; Waltham, MA, USA), denatured for 5 min at 65°C and added to a total of 20 μl of first‐strand cDNA synthesis reaction containing 1 U of RevertAid H Minus Reverse Transcriptase, 0.5 U of Ribolock, 4 mM dNTP mix and 1× RT buffer (ThermoFisher Scientific). The RT reaction proceeded at 42°C for 1 h, followed by 10 min at 70°C to deactivate the enzyme. qPCR reactions were carried out using PyroTaq EvaGreen mix Plus (ROX; CulteK Molecular Bioline, Madrid, Spain) according to the manufacturer's instructions. All analyses were performed in triplicate on an ABI 7500 Fast‐Real Time qPCR instrument (Applied Biosystems, Waltham, MA, USA). Specific oligonucleotides efficiencies were tested by qPCR using 10‐fold serial dilutions of the corresponding cDNA (Appendix Table S2 ). Expression analysis was performed by calculating the relative quantification (RQ) values with respect to the endogenous genes F‐BOX and PDF2 (Lilly et al, 2011 (link)) of three biological replicates. The Student's t‐test was applied to ΔCt values for statistical analysis.
7
First-Strand Synthesis Detailed Protocol
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First-strand synthesis was performed in a similar manner as described in Parkhomchuk et al (2009) (link). For first-strand synthesis, 8 μl fragmented mRNAs were mixed with 1 μl of random hexamers (Invitrogen) and 1 μl of 10 mM dNTPs. The samples were incubated at 65°C for 5 min and chilled on ice for 1 min. We added 10 μl of a master mix with final concentrations of 1× RT Buffer (Thermo Fisher Scientific), 10 mM MgCl2, 20 mM DTT, 4 U/μl RnaseOUT (Thermo Fisher Scientific), and 20 U/μl of SuperScript III RT (Thermo Fisher Scientific) to the RNA samples. The samples were then first incubated at 25°C for 10 min, followed by a 50-min incubation at 50°C. The reaction was stopped by incubating samples at 75°C for 15 min. For dNTP cleanup, 80 μl water, 1 μl glycogen, 10 μl 3 M NaOAc (pH 5.2), and 200 μl cold ethanol were added to the samples. Samples were stored at −80°C for 3–7 d. Samples were centrifuged at 14,000 rpm (Eppendorf Centrifuge 5424) for 20 min at 4°C. Supernatant was removed and 500 μl of cold 75% ethanol was added to the samples. Samples were centrifuged again at 14,000 rpm (Eppendorf Centrifuge 5424) for 10 min at 4°C. Supernatant was removed and samples were resuspended in a mixture composed of 51 μl RNAse-free water, 1 μl of 10× RT Buffer, 1 μl 100 mM DTT, 2 μl of 25 mM MgCl2. Second-strand synthesis was performed as described in Parkhomchuk et al (2009) (link).
8
Viral Genome Identification from Symptomatic Trees
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Total RNAs were isolated from 100 mg leaf tissue from 32 samples collected from symptomatic and non-symtomatic trees (Suppl. Table S1) using the extraction protocol described by Boom et al. (1990) . Pooled samples of three to five leaves from different twigs of each tree were used. Symptomatic trees exhibited most often mottle, or mottle in combination with a mixture of other symptoms like flecking, chrolotic ringspots, vein banding, chlorotic line pattern, mosaic and/or leaf deformation.
The first-strand cDNAs were synthesized from 1 µg of total RNA in a 20 µl reaction volume of 1 x RT buffer (Thermo Scientific) containing 1 µM dNTPs mix, 100 U RevertAid Premium reverse transcriptase (Thermo Scientific), 20 U Ribolock RNase inhibitor (Thermo Scientific) and 100 pmol of random hexameroligonucleotides (biolegio). Subsequent PCR amplifications were conducted in a 50 µl volume of 1 x DreamTaq Buffer (Thermo Scientific) containing 0.2 µM dNTP mix, 0.25 U of DreamTaq DNA polymerase and 1 µM of each forward and reverse primer. The designed primer pairs targeted regions of each of the identified viral genome segments (Table 1). The thermal cycles were as follows: 2 min at 94 °C followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, with a final extension step of 72 °C for 5 min. The product lengths amplified for the different RNA segments are shown in Table 1.
The first-strand cDNAs were synthesized from 1 µg of total RNA in a 20 µl reaction volume of 1 x RT buffer (Thermo Scientific) containing 1 µM dNTPs mix, 100 U RevertAid Premium reverse transcriptase (Thermo Scientific), 20 U Ribolock RNase inhibitor (Thermo Scientific) and 100 pmol of random hexameroligonucleotides (biolegio). Subsequent PCR amplifications were conducted in a 50 µl volume of 1 x DreamTaq Buffer (Thermo Scientific) containing 0.2 µM dNTP mix, 0.25 U of DreamTaq DNA polymerase and 1 µM of each forward and reverse primer. The designed primer pairs targeted regions of each of the identified viral genome segments (Table 1). The thermal cycles were as follows: 2 min at 94 °C followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, with a final extension step of 72 °C for 5 min. The product lengths amplified for the different RNA segments are shown in Table 1.
9
cDNA Synthesis from RNA with RT-PCR
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A 1-μg aliquot of freshly prepared RNA was reverse transcribed (RT) into cDNA using RevertAid (Fermentas), following the manufacturer’s instructions. The reaction mixtures contained 0.5 mM dNTPs, 5X RT Buffer (Fermentas), 1 u/μL RiboLock Ribonuclease inhibitor (Fermentas) and 25 ng/μL random hexamers (Promega). Prior to cDNA synthesis, RNA samples, random hexamers and dNTP’s were incubated at 60°C for 5 min. The reaction was carried out at 42°C for 60 min in a C1000 Thermal Cycler (BioRad, Munich, Germany). After that, the samples were heated to 70°C for 10 min to denature the enzyme, and then cooled to 4°C.
10
Total RNA Extraction and Reverse Transcription
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Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions, adding 5 μg glycogen as a carrier to facilitate RNA precipitation. 1 μg total RNA was subjected to a reverse transcriptase (RT) reaction in 25 μL reaction volume containing 1X RT-Buffer (Fermentas); 0.1 μg/μL bovine serum albumin (BSA, Sigma); 0.05% Triton X-100; 1 mM dNTPs; 7.5 μM random exanucleotide primers (Fermentas); 1 U/μL RIBOlock (Fermentas); and 200 U RevertAid M-MuLV reverse transcriptase (Fermentas). The reaction was performed for 10 min at 25°C, 90 min at 42°C, and 10 min at 90°C. Control reactions “RT-” (without the enzyme RT) and “H2O”, without RNA, were carried out.
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