Computational protocol: Collagen type IV at the fetal–maternal interface

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Protocol publication

[…] Ten proliferative endometrium, 13 secretory endometrium and 7 decidual samples were used to investigate the transcript levels of alpha1-6(IV) chains. These samples were dated as mid proliferative and mid secretory using Noyes' criteria and confirmed as consistent with the participant's reported last menstrual period and serum levels of progesterone and estradiol collected at the time of biopsy.The samples were flash frozen in liquid nitrogen and total RNA was extracted using Qiagen RNeasy Kit according to manufacturer's protocol. RNA samples were reverse transcribed to cDNA with the SuperScript VILO cDNA synthesis kit (Life Technologies, Invitrogen) using a mix of oligo(dT) and random hexamer primers according to manufacturer's instructions. qPCR experiments were carried out within three months after the synthesis of cDNA. Primers were designed using Geneious (Biomatters Ltd.) and the Primer-Blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Melting temperatures and potential formation of secondary structures were tested using OligoCalc (http://www.basic.northwestern.edu/biotools/OligoCalc.html). The primer specificity was confirmed by sequencing PCR products. Validated sequences are shown in . RT-PCR was carried out using Fast SYBR green mastermix and run on an ABI PRISM 7700 sequence detection system according to the manufacturer's instructions (Applied Biosystems, Warrington, UK) with following cycle conditions: 95 °C 20 s (1 cycle), 95 °C 3 s and 60 °C 30 s (40 cycles). A dissociation curve was derived at the end of each run to check specificity. Controls lacking template or reverse transcriptase were included to ensure no foreign or genomic DNA contamination during preparations. Reference genes hRPL-19 and 18s rRNA were used to normalise for the relative mRNA levels between samples. The normalisation factor was gained by calculating the arithmetic mean of the relative quantities of both reference genes . Samples were run in duplicates and standard curves for each target were prepared from pooled decidual samples. qPCR data was analysed using the Kruskal–Wallis and Dunn's multiple comparisons test. […]

Pipeline specifications

Software tools Geneious, Primer-BLAST, OligoCalc, Biotools
Applications Population genetic analysis, qPCR
Diseases Neoplasms