Total RNA was extracted from human kidney cells (HK2 cells) and complete UUO kidney samples using the Illumina’s Epicentre MasterPure Kit (Madison, WI, USA). Reverse transcription was performed for the miRNAs with MiRCURY LNA Universal RT Kit of Exiqon (Vedbaek, Denmark) and for the mRNA with High Capacity cDNA Reverse Transcription Kit of Thermo Scientific (Waltham, MA, USA) on a FlexCycler2 (Analytik Jena AG, Jena, Germany). Quantitative PCR amplification was performed on a StepOnePlus Real-Time PCR System (Thermo Scientific Waltham, MA, USA). Sybr Green technology was used for miRNAs according to Exiqon’s kit, while for the mRNA PCR the MESA BLUE qPCR MasterMix Plus kit from Eurogentec (Liege, Belgium). The primers used for the PCR are listed in Additional file 9 .
Mesa blue qpcr mastermix plus kit
Manufactured by Eurogentec
Sourced in Belgium
The MESA blue qPCR MasterMix Plus kit is a ready-to-use solution for real-time quantitative PCR (qPCR) analysis. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform qPCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Mesa blue qpcr kit, by Eurogentec (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mircury lna universal rt kit, by Qiagen (1 mentions)
Epicentre masterpure kit, by Illumina (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Flexcycler2, by Analytik Jena (1 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Dnase treatment, by Qiagen (1 mentions)
4 protocols using mesa blue qpcr mastermix plus kit
1
Quantitative Analysis of RNA Expression
2
RNA Extraction, Quantification, and RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA extraction was performed using Tri-Reagent according to the manufacture’s protocol. RNA concentration was determined using Nanodrop and 500–1000 ng of RNA was used to synthesise complementary DNA using Superscript II kit (Invitrogen). For RT-qPCR, the MESA Blue qPCR MasterMix Plus kit was used (Eurogentec).
3
Sciatic Nerve RNA Extraction and qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immediately after dissection, sciatic nerves were snap-frozen in liquid nitrogen and stored at −80°C until further processing. Nerves were crushed and homogenized on dry ice and then lysed in Trizol Reagent (Ambion). Nerves of three control or mutant animals were pooled for P5 and P15 analysis; nerves of two animals were pooled when the analysis was performed at 6 weeks. Cells were directly lysed in Trizol Reagent. RNA purification was performed using PureLinkTM RNA Mini kit (ThermoFischer Scientific). Manufacturer instructions were followed with an additional step of DNase treatment (QIAGEN) to degrade the genomic DNA. 500ng-1 μg/μl of RNA was then reverse-transcribed using Super-Script II Reverse Transcriptase (Invitrogen) and quantitative PCR (qPCR) was then performed using the MESA Blue qPCR Kit (Eurogentec). 5 μL of template cDNA and 20 μL of MESA blue qPCR MasterMix Plus kit (Eurogentec) including 100nM forward and reverse primers (see sequences in Table S1 ) were used per reaction in a 96-well plate. Water was used as a negative control. Relative expression values for each gene of interest were obtained after normalizing to b2m using the primers described in Table S1 .
4
Quantitative Analysis of Sciatic Nerve RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immediately after dissection, sciatic nerves were snap-frozen in liquid nitrogen and stored at -80°C until further processing. Both sciatic nerves of each animal were crushed and homogenised on dry ice and then lysed in Trizol Reagent (Ambion). One volume of 70% ethanol was added, and this solution was loaded onto Purelink micro columns (ThermoFisher). RNA was extracted following the manufacturer's protocol, including the DNAse digestion step, with all centrifugations performed at ≥12,000g for 15 seconds. 500ng-1μg of RNA was then reverse-transcribed using Super-Script II Reverse Transcriptase (Invitrogen), following the manufacturer's instructions. Quantitative PCR (qPCR) was then performed using the MESA Blue qPCR Kit (Eurogentec). 0.5μl of template cDNA and 12.5μl of MESA blue qPCR MasterMix Plus kit (Eurogentec) including 0.8μl of forward and reverse primers mix (at 5μM) (see sequences in key resources table) were used per reaction in a 96-well plate. Water was used as a negative control. Relative expression values for each gene of interest were obtained after normalising to B2M. Primer sequence of genes of interest can be found in the key resources table.
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!