Total RNA samples were analyzed using an Agilent Bioanalyzer RNA nanochip and 2ug arrayed into a 96-well plate. PolyA+ RNA was purified using the 96-well MultiMACS mRNA isolation kit (Miltenyi Biotec, Germany). The eluted PolyA+ RNA was ethanol precipitated and re-suspended in 10µL of DEPC treated water with 1:20 SuperaseIN (Life Technologies, USA). First-stranded cDNA was synthesized from the purified polyA + RNA using the Superscript cDNA Synthesis kit (Life Technologies, USA). The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol by replacing the dTTP with dUTP in dNTP mix, allowing second strand to be digested using UNG (Uracil-N-Glycosylase, Life Technologies, USA) in the post-adapter ligation reaction and thus achieving strand specificity. The cDNA was quantified using PicoGreen (Life Technologies, USA) and fragmented by Covaris E210 sonication. The paired-end sequencing library was prepared following the BC Cancer Agency Genome Sciences Centre strand-specific, plate-based and paired-end library construction protocol on a Biomek FX robot (Beckman-Coulter, USA). The 75 base PE libraries were sequenced on Illumina HiSeq2000 instruments. Analysis of mRNA expression was determined as previously described.(25 (link))
Multimacs mrna isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The MultiMACS mRNA isolation kit is a laboratory equipment designed for the extraction and purification of messenger RNA (mRNA) from various biological samples. It utilizes magnetic bead-based technology to efficiently capture and isolate mRNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Superase in, by Thermo Fisher Scientific (2 mentions)
Hiseq 2000, by Illumina (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Superscript cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Uracil n glycosylase, by Thermo Fisher Scientific (1 mentions)
Biomek fx robot, by Beckman Coulter (1 mentions)
Bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
E210 sonication, by Covaris (1 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Universal human reference total rna control, by Agilent Technologies (1 mentions)
Multimacs 96 separator, by Miltenyi Biotec (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
2 protocols using multimacs mrna isolation kit
1
Strand-Specific RNA Sequencing Protocol
2
Transcriptome Library Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The quality and yield of the isolated RNA (seven samples plus Universal Human Reference total RNA control (Agilent Technologies, USA)) was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). PolyA+ RNA was purified using the 96-well MultiMACS mRNA isolation kit on the MultiMACS 96 separator (Miltenyi Biotec, Germany) with on-column DNaseI treatment as per the manufacturer's instructions. The eluted PolyA+ RNA was ethanol precipitated and resuspended in 8 μl of Nuclease free water with 1:20 SuperaseIN (Life Technologies, USA). First strand cDNA was synthesized from the purified polyA+ RNA using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Synthesis of the second-strand cDNA was carried out in 50-μl reaction volume using second-strand reagents from SuperScript double-stranded cDNA synthesis kit (Life Technologies, USA). Double-stranded cDNA was quantified on Qubit Fluorometer using the Qubit dsDNA HS assay (Life Technologies, USA). The quality of cDNA was assesed on the Agilent Bioanalyzer using the high sensitivity DNA chip assay.
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!