A novel pcr enzyme formulation and qpcr detection reagents give utmost specificity. Sample to insight applicationnote purification of total rna from peripheral blood mononuclear cells steffen zingler and peter porschewski peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. The lunascript rt supermix kit requires the shortest reaction time and tolerates elevated temperatures, reducing complications from rna secondary structure. Simply isolate rna from your samples and convert to cdna using the rt. A control rna neo mrna is included in the cdna synthesis system to verify that the system is functioning correctly. Qiagen pcr arrays pathway focused products for research and development. The rt2 ht first strand kit procedure comprises 2 steps. The rt 2 easy first strand kit provides a rapid and convenient procedure for efficient cdna synthesis from mrna for use in gene expression assays. Control rtc tests the efficiency of the rt2 first strand kit c03 reaction with a primer set detecting the template.
The rt2 sybr green mastermix qiagen, valencia, ca was used for rtpcr, following the qiagen protocol for 384 well plates containing 4 replicates of 96 assays. First strand cdna was synthesized from 500ng total rna using the rt2 first strand kit qiagen according to the manufacturers protocol. Superscript iii firststrand synthesis supermix for qrtpcr. Review and cite pcr arrays protocol, troubleshooting and other. Rt2 profiler pcr array application examples download the. The realtime pcr modules transform threshold cycle ct values to calculated results for gene and mirna expression, somatic mutation detection and copy number measurements. Easy first strand kit provides a rapid and convenient procedure for efficient first strand cdna synthesis and genomic dna elimination in rna samples. Dna polymerases, dntps, pcrqpcr plastics consumables, pcr buffers and reagents. The synthesized cdna is ready to use in realtime pcr expression analysis of multiple genes.
From each group circa 400 ng of combined rna was used to prepare the corresponding cdna with the rt 2 first strand kit qiagen, germany and. Qiagen is the leading provider of innovative sample and assay technologies. Buffer ge 100 l, 5x buffer bc3 200 l, re3 reverse transcriptase mix 100 l, control p2 50 l, nucleasefree water 1 ml. First strand cdna synthesis protocols e6300 isolation of mrna using the polya spin mrna isolation kit. The geneglobe data analysis center is a complimentary resource for analyzing realtime pcr or ngs data. Intestinal ex vivo organoid culture reveals altered. When purchased with supertaq, this kit provides reagents, controls and protocols for reverse transcription and pcr. The ngs module supports the analysis of qiagen s qiaseq ngs library kits and target enrichment panels. For highspeed preparation of cdna without rna purification for use in realtime rtpcr.
Qiagen distributors please see the last page for contact information for your local qiagen distributor. Print bookmark share for pathwayfocused gene expression profiling using realtime rtpcr. For fifty 20 l first strand cdna synthesis reactions. Start by isolating total rna from your sample using the appropriate method. Technical guide to qiagen pcr arrays download the guide. For cdna synthesis and genomic dna elimination in rna samples for use with rt2 profiler pcr arrays and rt2 lncrna pcr arrays. Superscript iii firststrand synthesis system for rtpcr. The rt2 first strand kit provides a rapid and convenient procedure for efficient firststrand cdna synthesis and genomic. Primers do not proceed with the protocol until a primer for reverse transcription has been selected. Both oligodt and random primers for cdna priming are included, as is rnase inhibitor. I typically clean the rna by following the rna cleanup protocol from qiagen s rneasy mini kit. For pathwayfocused gene expression profiling using realtime rtpcr. Rtqpcr, rtpcr and cdna synthesis product listing application overview rna can be detected, quantified and manipulated through copying via the use of reverse transcriptases rts, which are rnadependent dna polymerases. The simple onestep reaction not only performs both polya tailing and reverse transcription at the same time.
The protocol describes the preliminary harvesting of bacteria and incubation with lysozyme to lyse their cell walls before dna purification. The kit includes a proprietary procedure to effectively eliminate contaminating genomic dna from rna samples before reverse transcription. Modified protocol for housekeeping gene pcr arrays 26 limited product warranty this product is intended for research purposes only and is not intended for drug or. Protocol for housekeeping genes rt2 profiler pcr arrays 55. Then, combine the template with an instrumentspecific and readytouse rt2 sybr green qpcr master.
Australia qiagen pty ltd po box 25 clifton hill victoria 3068 abn 75 072 382 944 orders 0394893666 fax 0394893888 technical 0394893666 canada qiagen inc. Page 3 first strand cdna synthesis using superscript. These are available online in convenient and compact pdf format at. Results with as little as 25 ng total rna per reaction. I also perform the optional on column dnase digestion provided within the qiagen kit to remove any. The complete pcr array system demonstrates strong correlations across technical replicates, lots, and instruments with average correlation. The first protocol describes firststrand cdna synthesis. Learn about the highspecificity agilent mirna qrtpcr detection kit and master mix detection kit. Invitrogen superscript ii reverse transcriptase is a genetically engineered mmlv reverse transcriptase rt with reduced rnase h activity and increased thermal stability compared to wildtype mmlv rt. The rt2 first strand kit provides a rapid and convenient procedure for efficient firststrand cdna synthesis and genomic dna elimination in rna samples. With the sensitivity of the rt 2 first strand kit, as little as 1 ng or as much as 5 g of total rna per array plate provides greater than 80% present call rates see figure positive results with as little as 25 ng rna reproducibility. Rt2 profiler pcr arrays enable gene expression profiling of hundreds of.
L 50 units and add sterile, distilled water to a 20. For cdna synthesis and genomic dna elimination in 96 rna samples for use with rt 2 profiler pcr arrays. The rt 2 mirna first strand kit contains all reagents needed for converting mirna into first strand cdna for realtime pcr. The rt 2 first strand kit is intended for molecular biology applications. Pcr polymerase chain reaction is used in a wide variety of applications including cloning, gene expression analysis, genotyping, sequencing, and mutagenesis.
The rt2 preamp cdna synthesis kit and rt2 preamp pathway primer mix are intended for molecular. The rt2 profiler pcr array system is the most reliable and accurate tool for. It also incorporates a proprietary and unique universal pcr priming sequence providing a scalable analysis solution for multiple mirna sequences in the same. I am planning to use the qiagen rt2 profiler pcr array for signaling. Troubleshooting and frequently asked questions 24 appendix. Rt2 profiler pcr array application examples download the article 1. Retroscript first strand synthesis kit for rtpcr from. Rna targets from 100 bp to 12 kb can be detected with this system. At just minutes, the lunascript rt supermix kit offers the shortest available first strand cdna synthesis protocol comparison of recommended protocols for cdna synthesis. Remove inhibitors by ethanol precipitation of mrna preparation before the first strand reaction.
Item rt2 first strand kit 50 company qiagen sample to insight. Superscript iii firststrand synthesis supermix for qrtpcr provides the hightemperature capability of superscript iii reverse transcriptase in an optimized supermix format for the synthesis of first strand cdna for use in realtime quantitative rtpcr qrtpcr. The protocol takes only two hours to perform per sample from start to finish. Kinase inhibition of g2019slrrk2 restores autolysosome.
The kits provide qualified reagents for polyadenylation of mirnas from total rna and synthesis of first strand cdna from tailed mirnas. Rt2 easy first strand kit 12 from qiagen selectscience. Can i use rt2 first strand kit qiagen with samples treated. Lysis and reverse transcription are performed in the same tube, and the resulting first strand cdna is ready to use in cloning and pcr. Pathwayfocused gene expression profiling with qrtpcr. I am planning to use the qiagen rt2 profiler pcr array for signaling pathways.
Mutations in the rnase h domain of the enzyme eliminate degradation of the rna during first strand. See figure 1 for an overview of the pcr array procedure. The rt2 ht first strand kit is intended for molecular biology applications. For best results, use the control rna by simply substituting your rna with 5. Purification of total rna from peripheral blood mononuclear cells download the application note 1. The rt 2 first strand kit provides a rapid and convenient procedure for efficient first strand cdna synthesis and genomic dna elimination in rna samples.
Is it really necessary to use the rt2 sybr green mastermixes and the rt2 first strand kit with the profiler array. Then, mix your template with one of our instrumentspecific. Rt2 first strand kit 50 from qiagen sample to insight. Next convert the experimental rna samples into pcr template with the rt2 first strand kit. For cdna synthesis and genomic dna elimination in rna samples for use with rt2 profiler pcr arrays. Mastermixes, and the rt2 first strand kit have been optimized in. Rt2 first strand kit 12, 330401 from qiagen labsave.
The thermo scientific pcr portfolio has everything you need for a successful pcr run. In my laboratory there is a rt 2 first strand kit to do the rtpcr to obtain cdna. This protocol is designed for purification of total dna from grampositive bacteria. Rt2 first strand kit 50 330404 from qiagen sample to.
9 208 865 1216 18 13 338 1507 100 1507 517 989 878 1342 612 766 1057 1122 1226 781 827 1247 1630 370 120 1374 645 124 329 474 1277 795 1369 13 937 852 441 315 853 524 689 1042