For qPCR, cells were allowed to grow to stationary phase under appropriate nutrient limiting environments in 10 ml culture volume. 5 ml of the culture was taken for RNA extraction. RNA extraction was done using the RNeasy Mini Kit (Qiagen) as per the manufacturer’s protocol. Extracted RNA was DNase treated using the Turbo DNA-free kit (Ambion) as per the manufacturer’s protocol. The DNA free RNA was run on a 1% gel for visual inspection. 500 ng of RNA (quantified using the Qubit RNA BR assay kit) was used for cDNA preparation using the High Capacity Reverse Transcription Kit (Applied Biosystems). RT-qPCRs were performed using the PerfecTa Sybr Green SuperMix (Quanta Biosciences). The house keeping genes used as reference in the analysis were cysG and hcaT. The transcript abundance for glnG and amtB was normalized to the geometrical mean of the levels of cysG and hcaT. Three biological replicates and 3 technical replicates were used in each case. The averages mentioned are based on biological replicates. Error bars represent standard deviation. Students-t test was performed to look for statistically significant differences.
Perfecta sybr green supermix
Manufactured by Quanta Biosciences
Sourced in United States, Moldova, Republic of
PerfeCTa SYBR Green SuperMix is a ready-to-use reaction mix for real-time quantitative PCR (qPCR) that contains all necessary components, including SYBR Green I dye, for the detection and quantification of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (26 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (15 mentions)
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Rneasy kit, by Qiagen (11 mentions)
Qscript cdna supermix, by Quanta Biosciences (11 mentions)
Qscript microrna cdna synthesis kit, by Quanta Biosciences (10 mentions)
Trizol, by Thermo Fisher Scientific (10 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (9 mentions)
Qscript mirna cdna synthesis kit, by Quanta Biosciences (7 mentions)
Iscript cdna synthesis kit, by Bio-Rad (7 mentions)
Cfx384 real time system, by Bio-Rad (7 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (7 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
M mlv reverse transcriptase, by Takara Bio (5 mentions)
Cfx96 real time system, by Bio-Rad (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (5 mentions)
Superscript 3, by Thermo Fisher Scientific (5 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (4 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
151 protocols using perfecta sybr green supermix
1
Quantitative PCR of Gene Expression
2
Quantitative Real-Time PCR Analysis
3
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated according to Trizol Reagent manufacturer’s instructions (Invitrogen). 9.0, 7.0, and 0.3 µg liver, kidney, and pituitary RNA, respectively, were subjected to PerfeCTa DNase I (Quanta BioSciences, Beverly, USA) digestion followed by reverse transcription in a final volume of 20 µL (qScript cDNA synthesis, Quanta BioSciences). For complementary DNA (cDNA) amplification, 1x PerfeCTa SYBR Green Supermix (Quanta BioSciences), cDNA, and 250 nM oligonucleotides (sequences are listed in Table S1 ) were mixed in a total volume of 10 µL following the thermal cycling program consisting of 3 min at 95 °C, followed by 41 cycles of 15 s at 95 °C, 20 s at 50 to 60 °C (for details see Supplementary Table S1 ), and 30 s at 72 °C. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using Mx3005P real-time PCR system (Stratagene, Agilent Technologies). Copy numbers were calculated based on standard curves. Expression levels were normalized to a composition factor based on the housekeeper genes hypoxanthine phosphoribosyltransferase 1 (Hprt), ribosomal protein L13a (Rpl13a), 18S ribosomale RNA, beta-actin, TATA-binding protein (TBP), and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) for liver and kidney samples or Hprt and Rpl13a for the pituitary glands.
4
Quantitative Analysis of IL-6 Expression
5
Real-Time qRT-PCR Analysis of miRNA and mRNA
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Real-time qRT-PCR analysis was performed as previously described11 (link)45 (link). Briefly, RNA was extracted using miRNeasy columns (Qiagen, Valencia, CA). miRNA expression analysis was performed using the qScript miRNA cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD) and PerfeCTa SYBR Green Supermix (Quanta Biosciences). miRNAs were amplified using specific mature miRNA sequences as forward primers and the universal primer provided in the kit as the reverse primer. U6 was used as internal control. A GeneAmp RNA PCR kit (Applied Biosystems, Carlsbad, CA) and POWER SYBR Green mix (Applied Biosystems) were used for mRNA quantification. PCR primer sequences are in Supplementary Table 3 .
6
Real-Time PCR Analysis of RNA and miRNA Expression
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Total RNA from mouse livers and HepG2 cells was extracted using TRIzol (Invitrogen), followed by DNase I treatment (Invitrogen), and reverse-transcribed using an Iscript cDNA kit (Bio-Rad). Real-time PCR was conducted using iTaq Universal SYBR Green Supermix (Bio-Rad) and the CFX connect ST system (Bio-Rad). The human and mouse primer list is detailed in Supplementary Table 5 . Normalization was performed against human or mouse β-actin and Gapdh. Pri-miR-22 expression was measured by real-time PCR on cDNA synthesized from total RNA, which was isolated as previously described.
MiRNA from total RNA extracted from mouse livers and HepG2 cells was purified using TRIzol, followed by an overnight ethanol precipitation. cDNA was synthesized using a qScript microRNA cDNA Synthesis kit (Cat# 95107; Quanta Biosciences, Beverly, MA, USA). MicroRNA expression was measured by real-time PCR using PerfeCTa SYBR Green Supermix (Cat# 95054; Quanta Biosciences). Normalization of miR-22–3p (PerfeCTa microRNA assay) was performed against human or mouse RNU6 (PerfeCTa microRNA assay). Total RNA was also used for in-house small RNA library preparation and miRNA sequence profiling as previously described [25 ]. The resulting FASTQ files were demultiplexed, mapped, and annotated using a published pipeline [26 (link)].
MiRNA from total RNA extracted from mouse livers and HepG2 cells was purified using TRIzol, followed by an overnight ethanol precipitation. cDNA was synthesized using a qScript microRNA cDNA Synthesis kit (Cat# 95107; Quanta Biosciences, Beverly, MA, USA). MicroRNA expression was measured by real-time PCR using PerfeCTa SYBR Green Supermix (Cat# 95054; Quanta Biosciences). Normalization of miR-22–3p (PerfeCTa microRNA assay) was performed against human or mouse RNU6 (PerfeCTa microRNA assay). Total RNA was also used for in-house small RNA library preparation and miRNA sequence profiling as previously described [25 ]. The resulting FASTQ files were demultiplexed, mapped, and annotated using a published pipeline [26 (link)].
7
Quantitative Analysis of Antimicrobial Peptide Expression
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Quantitative real-time PCR (qPCR) was used to assess rpRelish silencing on the expression of selected AMPs known to have a differential expression in fat body or hemocoel after infection with bacteria or protozoans. Primers were designed, or retrieved from the literature, for: α-Tubulin, rpRelish, rpCaspar, Prolixicin, Lysozyme-B, Defensin-A, and Defensin-C (S3 Table ). All reactions contained 4.4 μL of cDNA, 300mM of each primer, and 5 μL of PerfeCTa SYBR Green Super Mix (Quanta Biosciences, Gaithersburg, MD, US) in a final volume of 10 μL. qPCR was performed on a LightCycler96 thermal cycler (Roche diagnostics, Mannheim, Germany). The qPCR conditions used were: 95°C: 3 min, 40 cycles of 95°C: 15 s and 60°C: 30 s, followed by a melt curve analysis to confirm the specificity of the reaction. No-template controls were included with each primer set to verify the absence of exogenous DNA and primer-dimers. Relative differences in transcripts levels were calculated using the 2– ΔΔ Ct method [56 (link)–58 (link)] with α-Tubulin (RPRC003295) as the reference gene. PCR efficiencies (E) for each primer were determined using the slope of a linear regression mode.
8
Chromatin Immunoprecipitation Assay on Drosophila Embryos
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Embryos were collected for 30 min and then aged at 25°C for 110 or 170 min to target nc13 and nc14b developmental stages. Embryos were then fixed and manually sorted as previously described (21 (link)). The following volumes of antibodies were used in ChIP experiments: Pc, 10 μl (47 (link)); Pho, 5 μl (48 (link)); E(z), 10 μl (49 (link)); Pcl, 10 μl (50 ); Hb, 5 μl; Cad, 5 μl (21 (link)); H3K27me3, 0.2 μl (Millipore #07-449); H3, 0.5 μl (Abcam #ab1791); mock, 0.5 μl (immunoglobulin G, Cell Signaling #2729); H3K27ac, 0.2 μl (Abcam #ab4729); and P-Rpb1 CTD (S5), 2 μl (Cell Signaling #D9N51). To approximately equalize the chromatin amount per ChIP using embryos at different developmental stages, we used a 2:1 ratio of nc13:nc14b as determined by total mass. Carefully staged embryos were then subjected to ChIP assays, and the enrichment values were calculated as previously described (21 (link)). Quanta Biosciences Perfecta SYBR green supermix was used for qPCR. All amplifications were performed in triplicate using Rotor Gene RG3000 thermocycler (Corbett Research). Sequences of PCR primers are listed in table S2.
9
qPCR for Gene Expression Analysis
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For qPCR, cells were grown to OD600 = 0.3 (measured with 1 cm light path in a Shimadzu UV mini 1240 spectrophotometer) in 10 mL of either 0.1% glucose M9‐minimal media, 0.1% glucose M9‐minimal media supplemented with 0.1% of L‐isoleucine, or 0.1% glucose M9‐minimal media supplemented with 0.1% of L‐threonine. RNA was extracted from 500 μL of the cultures, using the RNeasy Mini Kit (Qiagen) as per the manufacturer's protocol, and DNase treated using the Turbo DNA‐free kit (Ambion) according to the manufacturer's protocol. The DNA‐free RNA was run on a 1% (w/v) agarose gel for visual inspection. Five hundred nanograms of RNA (quantified using the Qubit RNA BR assay kit) was used for cDNA preparation using the High Capacity Reverse Transcription Kit (Applied Biosystems). RT‐qPCRs were performed using the PerfecTa Sybr Green SuperMix (Quanta Biosciences). The house keeping genes used as references were cysG and hcaT. Three biological replicates and three technical replicates were used in each case. The averages are based on biological replicates. Error bars represent standard deviation. Two‐tailed Students‐t test was performed to test for statistically significant differences.
10
Reverse Transcription and Real-Time qPCR Protocol
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Reverse transcriptase reactions were performed using High Capacity cDNA Reverse transcription kit, (Thermo Fisher Scientific, cat. 4368814) according to manufacturer’s protocol using 300 ng of total RNA per 50 µl reaction. For each sample a “-RT” control reaction which did not contain the enzyme, was prepared to assess level of genomic DNA contamination. Real-time quantitative PCR was performed using RotorGene Q. All reactions were run in triplicate using PerfeCTa SYBR® Green SuperMix (Quanta Biosciences, cat. 95054–100), primers at 300 nM concentration each and 0.3 µl of cDNA in 10 µl reaction volume. The primers used are listed in Table 4 . Cycling parameters were 2 min at 95°C, then 45 cycles of 10 s at 95°C and 60 s at 60°C. Data was collected and analysed by RotorGene Q analysis software using ddCt method and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as a reference gene.
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