Gapdh primers

RNA was prepared using the RNeasy kit (Qiagen) and 2 µg of total RNA was used for cDNA preparation using Superscript Verso enzyme kit (Promega). cDNA product was amplified in a 10 µl reaction using SYBR-Green Supermix and standard gene-specific (MYC, CD133, GAPDH) primers (Applied Biosystems). All reactions were processed in a QuantStudio-3 PCR System and results analyzed by QuantStudio software (Applied Biosystems).

Total RNA was isolated from laser-captured sections of meibomian gland acini using the RNeasy Micro Kit (Qiagen Sciences, Germantown, MD, USA), and 1 μg of total RNA was used to make cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer's instructions. PCR reactions were performed with 0.8 μl cDNA using the Advantage 2 PCR kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and published CD147 primers.^{36 (link)} GAPDH primers (Applied Biosystems) were used as an internal control. Cultures of human corneal epithelial cells served as positive control. For CD147 amplification in laser-captured sections, the PCR reaction included an initial cycle of 95 °C for 5 min; 45 cycles of denaturation (95 °C for 30 s), annealing (64 °C for 30 s), and elongation (72 °C for 30 s); and a final extension cycle of 72 °C for 5 min. The PCR products (10 μl) were analyzed electrophoretically in 1% (wt/vol) agarose gels containing ethidium bromide and visualized under UV light. To verify the identity of the CD147 PCR product, the band in the agarose gel was excised, and the extracted DNA was sequenced at the DNA Core Facility of Massachusetts General Hospital, Boston, MA, USA.

Total RNA was isolated from ~20 mg of human skeletal muscle tissue using the acid phenol method [26 (link)]. Total RNA was isolated from C2C12 myotubes as previously described [27 (link)]. RNA quantity was confirmed with the NanoDrop (NanoDrop Technologies, Wilmington, DE, USA). ANT1, Rn18s and GAPDH primers were obtained from Applied Biosystems (Roche, Branchburg, NJ, USA). Real-time quantitative RT-PCR (qRT-PCR) reactions were performed as one-step reactions on the ABI PRISM 7900 real-time PCR system from Applied Biosystems (Nieuwerkerk aan den Ijssel, the Netherlands) as previously described [28 (link)]. SYBR Green was used as the reporter dye. For all assays performed in human samples, GAPDH was the reference gene, and for all assays performed on C2C12 myotubes, Rn18s was the reference gene. All expression data were normalised by dividing the target gene by the reference gene. All qRT-PCR measurements were performed in triplicate.

Isolated RNA (2 μg) (NucleoSpin RNAII kit; Macherey-Nagel, Bethlehem, PA) was reverse-transcribed (High Capacity Reverse Transcription Kit; Applied Biosystems, Foster City, CA) for real-time PCR (Taqman Gene Expression Master Mix, ALOX12 (HS00167524) and GAPDH primers; Applied Biosystems, Foster City, CA). All sample reactions were run in triplicate on the AB 7500 Fast Real Time PCR System. Relative expression of 12-LOX was quantified by the Ct value measured against the internal standard GAPDH using the 7500 Fast System SDS Software v1.4.0 (Applied Biosystems).

The cardiac expression of the mRNAs of two marker genes was investigated: ANP for cardiac hypertrophy and FBN for fibrosis.
Rodent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers and a probe with a VIC reporter dye were supplied by Applied Biosystems (product number 4308313). The ANP and FBN primers and FAM reporter dye probes were ordered from Eurofins MWG Operon: ANP probe 5′-TCGCTGGCCCTCGGAGCCTAC-3′, forward primer 5′-GAAAAGCAAACTGAGGGCTCTG-3′, reverse primer 5′-CCCCGAAGCAGCTGGAT TGC-3′, FBN probe 5′-TCGGAGCCATTTGTTCCTGCACGT-3′, forward primer 5′-TGT-AGGAGAACAGTGGCAGAAAGA-3′ and reverse primer 5′-CCGCTGGCCTCCGAA-3′.
The total RNA was extracted from the heart tissues stored in RNA Later by using the TRIzol (Invitrogen, Carlsbad, CA, USA) RNA extraction method [57 (link)]. Equal amounts of the isolated RNA were subsequently transcribed into cDNA by using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA) as per the manufacturer’s instructions. The iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) kit was applied to perform a qPCR. The mRNA expression was analyzed via the ΔCt method. The Ct values of the target genes (ANP and FBN) were normalized to that of GAPDH (reference gene) by using the equation ΔCt = Ct(reference) − Ct(target) and were expressed as ΔCt.