Relative gene expression was analyzed by real-time PCR using the 2-ΔΔCq method [17 (link)]. Amplification of mRNA was performed using a Rotor Gene 6000 instrument (Qiagen GmbH). For the real-time PCR reactions, microtubes (Qiagen GmbH) containing SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Inc.) were utilized. The following primers were used to amplify the candidate gene: COL6A1 sense, 5′-CGTGGACCTGTTCTTTGTG-3′, and COL6A1 antisense, 5′-CGTCACTGTAGTGCAGCG-3′. GAPDH, used for normalization, was amplified using the following primers: GAPDH sense, 5′-CATGTTCGTCATGGGTGTGA-3′, and GAPDH antisense, 5′-ATGGCATGGACTGTGGTCAT-3′.
Microtubes
Manufactured by Qiagen
Sourced in Germany
Microtubes are small, plastic containers used in various laboratory applications. They are designed to hold small volumes of liquids or samples securely during storage, transportation, and processing. Microtubes typically have a conical shape and are available in different sizes, usually ranging from 0.2 to 2.0 milliliters in capacity.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using microtubes
1
Relative Gene Expression Analysis by qPCR
2
Broth Aliquoting and DNA Extraction
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Culture broth from the above mentioned cultivation step was divided into aliquots for OD600 measurements, DNA extraction, and cryo-conservation using the VIAFLO 384 system (Integra Biosciences, Zizers, Switzerland). Glycerol stocks were prepared by first pre-filling tubes with 300 μL 80% glycerol using the Matrix Wellmate and then adding 200 μL of culture broth. For DNA extraction, 200 μL of broth was transferred to microtubes (Qiagen, Hilden, Germany) containing 2.3-mm zirconia beads (Carl Roth, Karlsruhe, Germany) and the cells were disrupted by 2 × 1 min pulses at 30 Hz using a TissueLyser II (Qiagen). The tubes were centrifuged for 2 min at 4,000 × g, incubated at 70°C for 45 min and centrifuged again as above. The supernatant was used for 16S rRNA gene amplification with primer pair E8F (5′-GAG TTT GAT CCT GGC TCA G-3′) and 1492R (5′-ACG GYT ACC TTG TTA CGA CTT-3′) (Lane, 1991 ).
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