Myometrial invasion classification: 10 cases in stage Ia, 16 case

Myometrial invasion classification: 10 cases in stage Ia, 16 cases in stage Ib and 6 cases in stage Ic. Patients were

also grouped according to the status of lymph node metastasis: 6 cases with lymph node metastasis and 26 cases free of lymph node metastasis. Methods RT-PCR technique to detect the expressions of Bcl-xl and Bcl-xs mRNA Total tissue RNA was extracted by following protocol provided PF299 in vivo in the TRIzol Crenigacestat reagent kit (DaLian TAKARA Biotechnology Company). The 1st strand of cDNA was synthesized according to protocol provided in the Reverse Transcription kit (Shanghai Invitrogen Biotechnology Co. Ltd.), while using a total of 15 μl of reaction system with 1.5 μl template RNA. The cDNA product was stored at -20°C for experiments. β-actin was included as an internal control and PCR assay was performed to amplify target genes. The volume of PCR reaction system was 25 μl: 3 μl template cDNA, 2.5 μl 10 × buffer, 2 μl 2.5 mM dNTP, 0.1 μl of each primers, and 0.2 μl 5 u/μl Taq-E and the total reaction volume was raised to 25 μl using deionized water. Bcl-xl primer sequences were: upstream 5′-GGCAACCCATCCTGGCACCT-3′, downstream 5′-AGCGTTCCTGGCCCTTTCG-3′, yielding predicted amplification

product of 472 bp. Bcl-xs primer sequences were: upstream 5′-GAGGGAGGCAGGCGACGAGTTT-3′, downstream 5′-ATGGCGGCTGGACGGAGGAT-3′, yielding predicted amplification product of 216 bp. β-actin primer selleck chemicals llc sequences were: upstream 5′-GTGGGGCGCCCCAGGCACCA-3, downstream 5′-CTCCTTAATGTCACGCACGATTTC-3′, yielding predicted amplification product of 498 bp. β-actin was used as internal control to normalize different reactions. PCR reaction was performed on an thermocycler (PTC-100™, USA). Amplification conditions for Bcl-xl were: initial denaturation at 94°C for 3 min, then proceeding with the following reaction conditions: a total of 35 cycles of denaturation at 94°C for 45 s, annealing at 59°C for 45 s, and extension at 72°C for 60 s before final extension at 72°C for 7 min. As for Bcl-xs, the process included: initial denaturation at 94°C for 3 min,

then proceeding with the following reaction conditions: a total of 35 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s, before final extension at 72°C for 7 min. 5 Acetophenone μl PCR product was subjected to 2% agarose gel electrophoresis (150 v) for 60 min and stained with ethidium bromide. RT-PCR amplification product was then observed under UV light. ΦX174Hinc II (TAKARA Co.) was included as the standard for relative molecular size. 1D KodaK image analysis software was used to observe and capture images. Optical density (A) ratio of target gene and β-actin RT-PCR amplification products was calculated to determine the relative mRNA content of the target gene. Western-blot assay to determine the expressions of Bcl-xl and Bcl-xs/l protein Cytosolic protein was extracted and sample OD values were determined by phenol reagent assay (0.305~1.254).

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