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Series GSE28660

Query DataSets for GSE28660

Status

Public on Mar 27, 2019

Title

Differential gene expression in uterosacral ligament from patients with recurrent and primary pelvic organ prolapse

Organism

Homo sapiens

Experiment type

Expression profiling by array

Summary

Objective: The objective of this study was to profile differential gene expression in three distinct patient groups.
Study Design: The groups consisted of women with recurrent POP, women with primary POP and women without POP. Whole human genome DNA microarrays were used to evaluate differential gene expression in uterosacral ligament (USL) tissues.
Results: 10 genes of interest were identified in USL. Eight genes were over expressed 30-fold or greater, and two genes were under expressed. Confirmation of differential gene expression used the technique of real-time RT-PCR. Five of the substantially over expressed genes functioned in the distribution, metabolism or deposition of adipose tissue. Homeobox genes D10 and D11 were under expressed.
Conclusions: The differential gene expression seen in the USL tissues in women with recurrent POP suggests that USLs are weakened by changes directed by these genes.

Overall design

Experimental factors: presence of disease (recurrent pelvic organ prolapse)

Experimental design: Three groups of human subjects were used for the study. Group1(n=4) were women undergoing surgery with no evidence or history of POP. Group 2 (n=5) were women undergoing surgical repair for primary POP. Group 3 (n=5) were women with recurrent and symptomatic POP undergoing surgical repair. USL tissue was obtained at surgery. Total RNA was extracted from USL tissue and processed for hybridization to Codelink Whole Human Genome microarrays.

Quality control steps: The cRNA that was synthesized from each USL was used for hybridization to a single CodeLink (Applied Microarrays) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions.

Samples used, extract preparation and labelling:
The origin of each biological sample: The samples were uterosacral ligament tissue from women.

Manipulations of biological samples and protocols used: The subjects were samples of convenience, patients undergoing surgery. Group1 were women undergoing surgery with no evidence or history of POP. Group 2 were women undergoing surgical repair for primary POP. Group 3 were women with recurrent and symptomatic POP undergoing surgical repair. The stage of POP was established by physical examination using the standard POP-Q scoring system.

Experimental factor: presence of recurrent pelvic organ prolapse vs. primary POP or no prolapse

Technical protocols: The USL samples were minced and soaked in 600 µl TRI reagent for one hour then homogenized in TRI reagent. The homogenization probe was rinsed in 400 µl fresh TRI reagent and the two aliquots of TRI reagent were mixed. Bromochloropropane and sodium acetate were added and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity of RNA were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip.

Labelled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 0.75 micrograms of total USL RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNA extraction columns. The concentration of cRNA was determined by spectrophotometry.

Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the CodeLink Whole Human Genome microarray slides, and hybridized for 18 hours at 37C.
The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

Keywords: pelvic organ prolapse, recurrent pelvic organ prolapse

Contributor(s)

Eyster KM, Fiegen M, Benson K, Van Eerden P, Hansen KA

Citation missing

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Submission date

Apr 15, 2011

Last update date

Mar 27, 2019

Contact name

Kathleen M Eyster

E-mail(s)

Kathleen.Eyster@usd.edu