RNA extraction and DNAse treatment were performed using GenElute™ Mammalian Total RNA Miniprep Kit and DNase70–On–Column DNAse I Digestion Set (Sigma−Aldrich®, Milan, Italy—as previously described [32 (link)]). Two µg of total RNA was reverse transcribed with a High–Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA, USA) in 20 µL of the reaction mix. For gene expression analysis, we used specific forward and reverse primers at the final concentration of 300 nM (Table 1 ), with 5 µL of cDNA, 10 µL of Bio–Rad SsoAdvanced Universal SyBR Mix, and topped to 20 µL with ddH2O. Real–Time PCR was performed in a Bio–Rad (Hercules, CA, USA) CFX Connect thermocycler running a custom program, which consisted of 95 °C for 30 s, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s.
Ssoadvanced universal sybr mix
Manufactured by Bio-Rad
The SsoAdvanced Universal SYBR mix is a ready-to-use solution for quantitative PCR (qPCR) experiments. It contains all the necessary components, including a modified hot-start DNA polymerase, dNTPs, and a SYBR Green I-based fluorescent dye, optimized for reliable and sensitive gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Ssoadvanced universal probe mix, by Bio-Rad (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Rnaeasy column, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
480 sybr green lightcycler pcr mix, by Roche (2 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Dnase70 on column dnase 1 digestion set, by Merck Group (1 mentions)
Cfx connect, by Bio-Rad (1 mentions)
Pcr topo 2, by Thermo Fisher Scientific (1 mentions)
Rq1 dnase 1, by Promega (1 mentions)
Cfx96 machine, by Bio-Rad (1 mentions)
Trizol reagent, by Promega (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first stand synthesis system, by Thermo Fisher Scientific (1 mentions)
7 protocols using ssoadvanced universal sybr mix
1
RNA Extraction, Reverse Transcription, and qPCR
2
Quantitative Real-Time PCR Protocol Guidelines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The minimum information for publication of quantitative real-time (MIQE) PCR experiments guidelines were followed throughout the collection of qPCR data (Bustin et al., 2009 (link)). Amplification of all genes was detected with SyBR Green dye which generates fluorescence based on the synthesis of double-stranded DNA. The reactions contained two µL of cDNA with 10 µL of Bio-Rad SsoAdvanced Universal SyBR Mix, 600 nM forward and reverse primer concentration, and topped to 20 µL with DEPC H2O. Each replicate of L. stagnalis tissue was subjected to qPCR reactions in triplicate. The qPCR reactions took place in a Bio-Rad CFX Connect thermocycler running a custom program. The custom qPCR program consisted of 95 °C for 30 s; 40 cycles of 95 °C for 15 s, 55 °C for 30 s. The plate was read by the machine to measure fluorescence at the end of each cycle.
3
Transcriptional Analysis of Borrelia opp Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated as previously described (91 (link)) from samples of 200 replete larvae, 150 flat nymphs, 20 replete nymphs, and ~5 × 107 DMC-cultivated spirochetes. cDNAs, prepared with and without reverse transcriptase, were assayed for opp gene transcripts using the primer pairs listed in Table S2 in the supplemental material. Optimized amplification conditions for each gene were determined using SsoAdvanced Universal SYBR mix (Bio-Rad, Hercules, CA). Expression of opp genes was determined using a TaqMan-based assay and SsoAdvanced Universal Probe Mix (Bio-Rad) and normalized to flaB transcripts (94 (link)). All assays were performed in quadruplicate with three biological replicates. Internal standards for each assay were generated by cloning the corresponding amplicon into pCR-TOPO 2.1 (Invitrogen) using designated gene-specific primers (Table S2 ) according to the manufacturer’s instructions.
4
qPCR Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA for qPCR was isolated with Invitrogen TRIzol reagent and then treated with Promega RQ1 DNaseI in accordance with the manufacturer’s instructions for both reagents. Reverse transcription for qPCR was performed with Invitrogen SuperScriptIII or SuperScriptIV after RNA was primed with a 1:1 mix of oligo dTs and random hexamers. qPCR was done on non-diluted or diluted cDNA (1/5x) with BioRad Sso Advanced Universal SYBR Mix and on a BioRad CFX96 machine. We quantified expression in 2–3 samples per sex across three technical replicates each (we tested 3 females and three males from B. germanica, and 2 females two males from both R. prolixus). Each sample was prepared with three technical replicates, the mean of which was taken for Ct value of each sample. Target gene expression was normalized to β-actin expression using the Δ Ct method (Schmittgen and Livak, 2008 (link)). A dilution series was used to test primer pairs for efficiency; only primer pairs with calculated efficiency between 90–110% were used.
5
Quantification of Xist Transcript Levels
6
Quantification of Xist Transcript Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In several experiments we determined the levels of Xist by RT-PCR. For experiments with ActinomcyinD treatment, the drug was dissolved in DMSO at 1mg/mL and added to the culture medium to a final concentration of 1μg/mL. For RT-PCR, cells were harvested in 1mL TRIzol (Thermo Fisher), after culture medium removal and PBS wash. RNA was purified over RNAeasy columns (Qiagen). 1μg total RNA was used in a reverse-transcription (RT) reaction with SuperScript III and appropriate strand-specific reverse primer, according to manufacturer’s instructions (ThermoFisher). 1/20th of the RT reaction was used in a quantitative PCR reaction, using either 480 SYBR Green LightCycler PCR mix (Roche), SsoAdvanced Universal SYBR mix (Bio-Rad) or SYBR Green Master Mix (Applied Biosystems) and appropriate primers (see Supplementary Table 1 ), in triplicate reactions. RT-qPCR experiments were normalized against Gapdh or Rrm2 transcripts.
7
Quantification of oppA Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers used for qRT-PCR assays are listed in S2 Table . Total RNA was isolated as previously described [114 (link)] from triplicate cultures of temperature-shifted spirochetes at late logarithmic phase of growth. cDNAs were generated with and without reverse transcriptase using the SuperScript III First Stand Synthesis System (ThermoFisher Scientific). qRT-PCR assays were developed to measure oppA1 and oppA2 transcripts in the tn mutants by flanking the transposon insertion site in each gene (Fig 1A ). oppA transcripts were quantified using SsoAdvanced Universal SYBR Mix (Bio-Rad, Hercules, CA) and normalized to flaB transcripts [115 (link)] (S2 Table ) using SsoAdvanced Universal Probe Mix (Bio-Rad). All assays were performed in quadruplicate with three biological replicates. Generation of internal standards for each assay were previously described [14 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!