KXK carried out the study on Reissner's membrane, including microdissection of tissues, primer design and validation, RNA isolation, quantitative analyses of PCR, preliminary patch clamp recordings and contributed to writing the manuscript.
DY and KN carried out the study, including microdissection of tissues, primer design and validation, RNA isolation, quantitative analyses of PCR, protein isolation, all steps in the immunoblots and their analysis, preparation of ears for immunohistochemistry and contributed to writing the manuscript.
Because of the presence of the AS CA9 transcript in the normal and/or non-hypoxic tissues, primers or probes designed for detection of the regions that are not affected by splicing cannot differentiate between the two forms of CA9 mRNA and thus might give false-positive results, which could influence the real clinical value of the hypoxia-induced FL CA9.
Full methodological descriptions of the tissue processing, primer sequences, in situ hybridization, and quantification are available in Hackett et al. (2015, in press) [ 53].
An additional non-normalized cDNA library was generated for the Heliothis virescens pheromone gland tissue by primer extension with the MINT cDNA synthesis kit (Evrogen) according to the manufacturer's protocol.
A fragment of RPS was PCR amplified from bisulfite treated genomic DNA, extracted from protonema tissue, using primers RPS-top-R-new and RPS-top-F (primers listed in Supplementary Table 3) and KAPA HiFi Uracil + polymerase (kappa biosystems), then cloned into pJET1.2 (Thermo Fisher Scientific).
This novel isoform was detected in 1/62 subclones from brain, and amplified at low levels throughout the nervous system and embryonic tissues, with primer pairs DIC1_Ex 1 for and DIC1_R rev, and DIC1_Ex 1 for and DIC1_iso14 rev, but it does not amplify in other tissues.
Probes were made by PCR amplification from human fetal tissue using primers as listed in supplementary material Table S1.
Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control gene was analyzed in cDNA of all tissues (forward primer: 5'-TACATGGTCTACATGTTCCAGTATG-3', and reverse primer: 5'-CAGTCTTCTGGGTGGCAGTGATG-3', amplicon: 440 bp).
Additional reactions were performed with control tissues and primer pairs located in varying parts of the predicted transcripts, in order to determine which exons are present in the mature mRNA (Table 1; APITD1-Expr 1 6).
To specifically amplify Rap1A retrogenes from Rap1A -/- tissues one primer was set in the targeted second exon: 5'-TCAGGAGGCGTGGGGAAG-3' and the other in the 3' UTR: 5'-GCCATAGAAATCAGTTATCCC-3'.
