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))
Uracil n glycosylase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Uracil-N-glycosylase is an enzyme that catalyzes the removal of uracil residues from DNA. It is commonly used in molecular biology applications to prevent carryover contamination in PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Taqman universal master mix 2, by Thermo Fisher Scientific (6 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (4 mentions)
Taqman universal pcr master mix 2, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000, by Illumina (2 mentions)
Abi prism 7900ht instrument, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Multimacs mrna isolation kit, by Miltenyi Biotec (1 mentions)
Superscript cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Biomek fx robot, by Beckman Coulter (1 mentions)
Bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
E210 sonication, by Covaris (1 mentions)
7900ht system, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
Pcr buffer, by Quanta Biosciences (1 mentions)
15 protocols using uracil n glycosylase
1
Strand-Specific RNA Sequencing Protocol
2
Quantification of MAPT Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One µg of total RNA was used to generate cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon sur Yvette, France) with Multiscribe Reverse Transcriptase and random primers. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA). To quantify total MAPT mRNA levels, we used the Hs00902194_m1 probe located at the junction between exons 12 and 13 (i.e. within a region present in all coding transcripts). To assess the cDNA expression of transcripts from promoter A, we designed a custom qPCR TaqMan® assay with a probe overlapping exons 0 and 1. UBC (Hs00824723_m1) was used as a control housekeeping gene. For each sample, all qRT-PCRs were performed in triplicate on an ABI PRISM® 7900 HT instrument (Applied Biosystems), according to the manufacturer’s protocol. The comparative CT method (2−ΔΔCT) was used to calculate relative mRNA expression levels.
3
RNA-Seq Library Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA samples (2 µg) were arrayed into 96-well plates, and polyadenylated mRNA was purified with a MultiMACS mRNA isolation kit as per the manufacturer’s instructions. First-strand cDNA was synthesized using a SuperScript cDNA Synthesis kit with random hexamer primers. The SuperScript cDNA Synthesis protocol was used for second-strand cDNA synthesis. dTTP was replaced with dUTP in the dNTP mix which allowed the second strand to be digested with UNG (Uracil-N-Glycosylase, Life Technologies, USA) in the post-adapter ligation reaction. The cDNA was quantified and checked for quality before fragmentation. Plate-based libraries were created following the BC Cancer Agency’s Michael Smith Genome Sciences Centre (BCGSC) paired-end (PE) protocol36 (link). The libraries were sequenced using Illumina HiSeq 2000 or 2500, 2 × 100 PE lanes, with v3 chemistry and HiSeq Control Software version 2.0.10.
4
Quantification of MAPT mRNA Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each of the 90 RNA samples, 1 µg of total RNA was used to generate cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Saint Aubin, France) with Multiscribe Reverse Transcriptase and random primers. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, with Uracil-N glycosylase (Life Technologies, San Francisco, CA, USA). TaqMan® assays were used to quantify mRNA levels of MAPT (Hs00902194_m1) using UBC (Hs00824723_m1) as the control housekeeping gene. All qRT-PCR were performed on an ABI PRISM® 7900 HT instrument (Applied Biosystems™) in triplicate for each sample, according to the manufacturer's protocol. The comparative CT method (2 - ΔΔCT ) was used to calculate the relative expression levels.
5
Quantification of Let-7 MicroRNA Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two steps of Real-time PCR were performed on 20 ng of transcribed total RNA using the TaqMan MicroRNA Reverse Transcription kit and TaqMan® Universal PCR Master Mix II, without uracil N-glycosylase (Thermo Fisher Scientific), according to the manufacturer’s instructions. TaqMan microRNA assays (assay ID): hsa-let-7a (000377), hsa-let-7b (002619), hsa-let-7c (000379), hsa-let-7d (002283), hsa-let-7e (002406), hsa-let-7f (000382), hsa-let-7g (002282), hsa-let-7i (002221), hsa-miR-98 (000577), and cel-miR-39-3p (000200) were used in the assay. All PCR reactions were performed on the 7900HT system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative expression level of each Let-7 member was determined using the comparative CT method, which was defined as 2−ΔCt, where ΔCt = Ct of the Let-7 member—Ct of cel-39 as described previously [11 (link)].
6
Quantitative Protein Detection by PLA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human IL8 was serially diluted in PLA buffer (1 mM D-biotin (Life Technologies), 0.1% BSA (Sigma-Aldrich), 0.05% Tween 20, 100 nM goat IgG (Sigma-Aldrich), 0.1 µg/µl salmon sperm DNA (Life Technologies), 5 mM EDTA in PBS), or in either 10% or 50% chicken serum prepared in PLA buffer. Individual dilution series included a negative control with no spiked antigen. Two µl of each sample were mixed with 2 µl of 60 pM PLA probes (either purified anti-IL8-Arm1_long and anti-IL8-Arm2_long conjugates, unpurified anti-IL8-Arm1 and anti-IL8-Arm2 conjugates, or a pair of purified conjugates with spiked-in anti-IL8 or Arm1 and Arm2, respectively) and incubated for 1.5 h at RT. After incubation, 1 µl of the mixture was transferred to 25 µl ligation and quantitative PCR mixture (1× PCR buffer (Quanta Biosciences), 2.5 mM MgCl2 (Quanta Biosciences), 0.5× Sybr Green I (Life Technologies), 0.1 µM BioFwd primer, 0.1 µM BioRev primer, 0.025 µM BioSplint, 0.08 mM ATP (Thermo Scientific), 0.2 mM dNTPs (with dUTP) (Thermo Scientific), 0.03 U/µl AccuStart Taq DNA polymerase (Quanta Biosciences), 0.01 U/µl T4 DNA ligase (Thermo Scientific) and 0.002 U/µl Uracil N-glycosylase (Thermo Scientific)). Quantitative PCR was performed in an MX3005 cycler (Stratagene) with an initial incubation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.
7
Fecal DNA Extraction and Quantitative PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total DNA was extracted from approximately 100 mg of feces by using the Zymo fecal DNA miniprep kit according to the manufacturer’s instructions (Zymo Research). Negative controls for detection of kit contamination were included and failed to produce visible PCR bands in an agarose gel but were analyzed as quality controls. Quantitative PCR was carried out using standard curves of known cultures prepared by serial dilution and extracted in the same manner as the fecal samples. Individual samples were analyzed in duplicate under the conditions listed in Table 5 . Reactions were carried out in 20-µl volumes with PerfeCTa SYBR green FastMix ROX (QuantaBio; Beverly, MA USA) or TaqMan universal master mix II, with uracil-N-glycosylase (Thermo Fisher Scientific; Waltham, MA, USA), in 5 µl of extracted DNA in a QuantStudio 3 qPCR machine (Thermo Fisher Scientific; Waltham, MA, USA).
8
Quantitative Gene Expression and ChIP Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated with the Qiagen RNeasy Mini Kit. Equal amounts of cDNA were synthesized using a High Capacity Reverse Transcription kit (ThermoFisher). TaqMan primers were mixed with TaqMan Universal Master Mix II, with uracil N-glycosylase (ThermoFisher). 18S was amplified as an internal control. QPCR was performed using the Bio-Rad CFX connect system with TaqMan Primer Assays to detect 18S and target genes (ThermoFisher). Primer pair sequences used to detect input and immunoprecipitated DNA sequences for ChIP assay were 5’-CCTGAGAATTGATGGGGAAA and 5’-GTGAACAGGTACCGCACAGA from -329 to -99 of the 5’-flanking RFX6 promoter and 5’-TGACCGGCTTGTCCTTAAAC and 5’-TGAATGGACGGACACTGGTA from -625 to -411 of the 5’-flanking mouse MCT1 promoter.
9
Quantifying miRNA Expression in Leiomyoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from leiomyoma and matched myometrial tissues to estimate miR expression levels using the miRVana RNA Isolation Kit (Ambion) according to the manufacturer's instructions. The RNA was eluted in 30 µL of nuclease-free water. The concentration of the extracted RNA was assessed using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and 10 ng was used to generate cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems by Thermo Fisher Scientific, Baltics, Lithuania). Quantitative real-time polymerase chain reaction (qRT-PCR) for the targeted miRs was performed using TaqMan Universal Master Mix II, uracil-N-glycosylase (UNG) (Applied Biosystems), and sets of miR-139-5p and U6 small nuclear RNA (U6 snRNA) (Applied Biosystems). All RT-PCR reactions were performed on a 7300 RT-PCR system (Applied Biosystems) for 40 amplification cycles; each reaction was performed in triplicate. Threshold cycle (Ct) and melting curves were estimated using the 7300 software program (Applied Biosystems). Relative miR levels were calculated using the comparative Ct method and normalized to U6 levels.21 (link)
10
DMRT3 Gait Allele Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Previously published TaqMan™ SNP genotyping assays for the DMRT3 lateral gait associated allele were utilized for gait analysis [35 (link)] in the ENH and the 8 related breeds (Group A). Of the latter, 6 breeds (Finn Horse, Konik, Gotland Pony, Icelandic Horse, Norwegian Fjord, and Shetland Pony) have already been genotyped for the DMRT3 gait allele [36 (link)]. Therefore, we genotyped the remaining 3 breeds for the gait mutation in a total of 114 horses: 33 ENH, 52 Hucul Horses, and 29 Exmoor Ponies. We used a CFX-96 Real Time-PCR machine (Bio Rad, Hercules, CA, USA) and corresponding software for PCR amplifications, genotyping and allelic discrimination. The thermal conditions were: priming at 60 °C for 1 min, initial denaturation at 95 °C for 10 min, 40 cycles at 92 °C for 15 sec each, annealing at 60 °C for 30 s, and extension at 60 °C for 1 min, followed by a final extension at 65 °C for 10 min. The 8 µL reactions contained 0.208 µL of TaqMan™ assay, 30 ng template DNA and 4.2 µL of ABI TaqMan Universal Master mix, no uracil-N-glycosylase (UNG) (Applied Biosystems).
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!