Real-Time Fluorescent Quantitative Polymerase Chain Reaction Study of Polo-Like Kinase-1 in Pterygia

Abstract

This study was designed to examine the expression of polo-like kinase 1 (PLK1) mRNA in 16 pterygia and 13 normal conjunctival tissue specimens using real-time fluorescent quantitative polymerase chain reaction (PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The difference in threshold cycle value (DeltaC(t)) was derived for PLK1 and GAPDH for each sample assayed, and the difference between the paired samples (DeltaDeltaC(t)) was calculated. The mean +/- SD DeltaC(t) of PLK1 mRNA was 9.56 +/- 1.30 in pterygia compared with 10.71 +/- 1.39 in normal conjunctiva. The expression of PLK1 mRNA in pterygium was 2.08 - 2.36 times that in normal conjunctiva; this difference was statistically significant. Real-time fluorescent quantitative PCR analysis appears to be effective and sensitive when determining the level of PLK1 mRNA expression. Using this method, it was demonstrated that PLK1 mRNA is over-expressed in pterygia, indicating a probable role for PLK1 in their development.

Transcriptome Analysis of Pterygium and Pinguecula Reveals Evidence of Genomic Instability Associated with Chronic Inflammation

“…The first group comprised genes involved in mitotic checkpoints and DNA repair. Differential expression of some DNA repair genes in pterygium vs. conjunctiva has been reported in previous gene expression profiling studies [ 44 , 45 ]. The second group comprised genes involved in inflammation and the immune response.…”

“…It was previously reported that genes involved in double-strand DNA break repair, RAD50 , RAD51 , XRCC2 and XRCC3 , are differentially expressed in pterygium [ 44 ]. In addition, PLK1 , which regulates the activity of RAD51, was identified as highly expressed in pterygium [ 45 ]. We already had XRCC2 listed; we added RAD50 , RAD51 , RAD51B , RAD51C , RAD51D , and XRCC3 .…”

Knockdown of DEPTOR inhibits cell proliferation and increases chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting PI3K/AKT activity

“…From control shRNA-treated RPMI-8226 cells, parental RPMI-8226 cells and DEPTOR shRNA-treated RPMI-8226 cells, total RNA from 1 × 10 6 cells was extracted using TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions and the method of Lu et al. 13 Total RNA (4 µg) was reverse transcribed to cDNA using the ThermoScript™ RT–PCR System Kit (Gibco) according to the manufacturer’s instructions. Primer sequences for RT–PCR for the human DEPTOR were 5′-CCTACCCAAACTGTTTTGTCGC-3′ (sense) and 5′-CGGTCTGCTAATTTCTGCATGAG-3′ (antisense).…”

“…From control shRNA-treated RPMI-8226 cells, parental RPMI-8226 cells and DEPTOR shRNAtreated RPMI-8226 cells, total RNA from 1 Â 10 6 cells was extracted using TRIzol Õ reagent (Invitrogen) according to the manufacturer's instructions and the method of Lu et al 13 Total RNA (4 mg) was reverse transcribed to cDNA using the ThermoScript C for 3 min, followed by 35 cycles of denaturation at 95 C for 15s, annealing at 60 C for 30s, and elongation at 72 C for 45s, followed by a final elongation step at 72 C for 10 min. The transcript levels were normalized according to the GAPDH transcripts.…”

Effect of β-(3,4-Dihydroxyphenyl) Lactic Acid on Oxidative Stress Stimulated by High Glucose Levels in Human Peritoneal Mesothelial Cells

“…Total RNA was extracted from 1 × 10 6 HPMCs using TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions and the method of Lu et al 15 Firststrand cDNA was synthesized from 2-µg aliquots of total RNA by reverse transcription (RT) using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Glen Burnie, MD, USA). Real-time quantitative polymerase chain reaction (PCR) was performed using the Taq 2× PCR MasterMix kit (Tiangen Biochemical Technology, Beijing, China) with SYBR ® Green-based detection chemistry (Invitrogen), on a LightCycler ® Run 5.32 Real-Time PCR System (Roche, Berlin, Germany; 1 µl of cDNA was used in each PCR).…”