For chicken neural crest RNA-Sequencing, we FACS isolated at least 5,000 cranial neural crest cells into the lysis buffer from the RNAqueous micro kit (ThermoFisher #AM1931). RNA was extracted according to the manufacturer’s protocol, quantified using a Qubit RNA HS Assay (ThermoFisher #Q32852), and analyzed for quality on an ABI 3730×l DNA Analyzer. RNA-Sequencing libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB #E7765) according to the manufacturer’s protocol. Depending on the input RNA amount, libraries were PCR amplified 13–16 cycles. Libraries were quantified using a Qubit DNA HS Assay (ThermoFisher #Q33230) and checked for fragment size distribution and quality on an ABI 3730×l DNA Analyzer. Individual samples were pooled at an equimolar ratio calculated using the KAPA Library Quantification Kit (Roche #07960336001) and sequenced in a single-end configuration on an Illumina NextSeq500 using the High Output 75bp kit. Sequencing was performed by the Biotechnology Resource Center (BRC) Genomics Facility (RRID:SCR_021727) at the Cornell Institute of Biotechnology.
Am1931
Manufactured by Thermo Fisher Scientific
Sourced in Belgium, United States
The AM1931 is a laboratory instrument designed for performing analytical tasks. It features high-precision measurements and reliable performance. The core function of the AM1931 is to provide accurate and consistent results for laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
Qubit dna hs assay, by Thermo Fisher Scientific (1 mentions)
Qubit rna hs assay, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Sds v2, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system thermocycler, by Bio-Rad (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Lmd6500 system, by Leica (1 mentions)
4 protocols using am1931
1
Transcriptome Profiling of Chicken Neural Crest
2
BDNF Expression in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation and reverse transcription reaction were performed as previously described in Kim et al. (2019 (link)). RNA extraction was done from p9 whole brain tissue of WT and BDNF+/– mice using RNAqueous kit with no alterations to procedure (AM1931, Thermofisher). qPCR was executed using 7900HT Fast Real-Time PCR system (Applied Biosystems), data were analyzed using SDS v2.4 (Applied Biosystems). GAPDH was used as a reference housekeeping gene. Delta (Δ) CT (MeanGene- MeanGAPDH) was utilized to calculate ΔΔCT (ΔCTPos-ΔCTNeg), and data was normalized to WT (100%). Mouse primers used: BDNF Forward: 5’-TCGTTCCTTTCGAGTTAGCC, BDNF Reverse: 5’-TTGGTAAACGGCACAAAAC, GAPDH Forward: 5’-AGTATGACTCCACTCACGGCAA, and GAPDH Reverse: 5’-TCTCGCTCCTGGAAGATGGT.
3
Acinar Cell Isolation and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acinar cells were isolated from pancreata of Ela-KrasG12D/+ mice by FACS, as previously described [28 (link)]. RNA was extracted using a column-based approach (AM-1931, ThermoFisher Scientific, Merelbeke, Belgium). The RNA integrity number (RIN) was measured using Agilent 2100 Bioanalyzer and samples with RIN > 7 were selected for experiments. Reverse transcription was performed on 150 to 250 ng of total RNA using the M-MLV reverse transcriptase (M1705, Promega, Leiden, Netherlands). Quantitative PCR was carried out in a final volume of 10 µL (1 µL neosynthetized cDNA, 2 µL primers 10 µM, 5 µL Sybergreen KAPA mix 2× (KK4601, Sigma Aldrich, Overijse, Belgium), 2 µL nuclease-free water) using the CFX96 Real-Time System thermocycler (C1000, Biorad, Temse, Belgium). The expression of target genes was normalized to the Rpl04 housekeeping gene. Relative expression was calculated using the ΔΔCt method. Primers used in the study are listed in Table 2 .
4
Laser Microdissection of Purkinje Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The slides were microdissected with the LMD6500 system (Leica) as already described by Pieczora et al. [41 (link)] using the following settings: 40× objective, power 23, aperture 21, speed 23 and specimen balance 18. With each session, about 1,000,000 µm2 of PCs was lasered and collected in non-adhesive 0.5 mL tubes by marking PC separately for the laser beam. After microdissection, 40 µL of lysis solution (AM1931; Thermo Fisher, Waltham, MA, USA) was added to each sample. All samples were stored at −80 °C until further usage. In total, about 4,000,000 µm2 of PC from four different rat cerebella were collected for each experimental group (1–3).
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!