The association between telomerase activity and expression of its RNA component (hTR) in breast cancer patients: the importance of DNase treatment

Telomerase is a ribonucleoprotein enzyme that compensates for the telomere length shortening which occurs during the cell cycle. Telomerase activity has been detected in most tumours but not in somatic cells. However, hTR; the RNA component of telomerase; has been reported to be universally expressed in both cancerous and non-cancerous tissues. Tumour samples from 50 patients with primary invasive breast cancer were collected. The TRAP assay was used to detect telomerase activity. RT-PCR on cDNA and DNased cDNA samples and control groups was used to detect the expression of hTR, GAPDH and PGM1 genes. Seventy-two percent of samples showed telomerase activity. DNA contamination was detected in 36 (72%) of RNA samples. Without performing DNase treatment, 49 (98%) of all samples showed hTR expression, but with the application of this strategy, hTR expression decreased from 98% to 64%. A significant association (p < 0.001) between hTR expression and telomerase activity was observed. Among the 32 hTR positive samples, 30 had telomerase activity and among the 18 hTR negative samples, telomerase activity was observed in 6 cases. Thus the application of this strategy could provide an applicable tool to use instead of the TRAP assay thus facilitating telomerase research in cancer genetic investigations.

The RNA component of telomerase acts as the template for telomeric repeat synthesis. In man, hTR is transcribed by RNA polymerase II and its mature transcript consists of 451 nucleotides [16]. The hTR gene was cloned and local-ized to chromosome 3q26. 3. in 1998 [17,18]. The template for hTERT activity lies in nucleotides 46 to 53. Although there is a variation of hTR RNA sequences among telomerase RNAs, there is a remarkably conserved secondary structure from ciliates to vertebrates. This indicates an essential role for the structure in enzyme function [19]. hTR is a single-copy gene that lacks poly A and does not contain any introns, so RT-PCR for hTR gene is considered to be prone to errors. DNA contamination of RNA extractions could be amplified by PCR and therefore could give rise to a false positive result for hTR transcription affecting the correlation between hTR expression and telomerase activity [20,21]

Patients
Institutional guidelines including ethical approval and informed consent were followed. We investigated 50 tumour samples from patients with primary invasive breast cancer treated surgically during 2004-2005 at Tehran University of Medical Sciences. Breast tissues were collected and preserved by rapid freezing in liquid nitrogen immediately after surgical excision and then were stored at -70°C.

The TRAP assay
Telomerase activity was analyzed using the PCR-based telomeric-repeat amplification protocol (TRAP assay) as previously described [22].
The PCR products were mixed with a loading dye and electrophoresed on 10% non-denaturing polyacrylamide gel, and finally, stained by silver nitrate which has more sensitivity than SYBR Green and Ethidium bromide.
An internal control was used to detect false-negative samples containing PCR inhibitors (such as CHAPS). In order to avoid false-positive results (characterized by variation in band intensity), negative controls were used. It was produced by heating the protein extract for 5 min at 90-95°C (to remove the enzyme activity) or using any telomerase negative sample (Fig. 1).

RT-PCR analysis
Total RNA was isolated from samples using Tripure Isolation Reagent (Roche, Germany). One microgram of total RNA, with random hexamer and M-Mulv reverse transcriptase enzyme (Fermentas co, Canada.) were used to create cDNA for each sample, according to the manufacturer's protocol. In order to avoid he probable DNA contamination for RNA samples, the following stages were performed. We prepared a solution containing the same Telomerase activity detected by TRAP assay method Figure 1 Telomerase activity detected by TRAP assay method. C.-: A negative control group including a telomerase negative sample, a denatured protein sample and a mixture without protein extract. materials used for cDNA synthesis excluding reverse transcriptase enzyme (negative control 1). This product contains DNA only, with a new concentration similar to the cDNA products, however, in the PCR products of these samples, the presence or absence of any DNA contamination could be observed and detected.
In order to perform DNase treatment, 1 µg of total RNA was digested by DNaseI (Fermentas Co, Canada) according to the manufacturer's protocol. Half of DNase treated RNA sample was used to create cDNA. The remaining half of the sample contained all of the materials excluding the reverse transcriptase enzyme (negative control 2), in order to validate the accuracy of DNase treatment process.
The cDNA, DNase treated cDNA and two control group samples were amplified in a 25 µl reaction mixture containing 0.2 µM of each primers and 1U Taq DNA polymerase (Fermentas co., Canada). GAPDH was amplified using GapF (5'-GGGAAGGT GAAGGTCGGAGTC-3') and GapR (5'-AGCAGAGGGGGCAGA GATGAT-3') oligonucleotides with an initial heating at 95°C for 3 min, followed by 30 cycles of 94°C for 45 s, 63°C for 45 s, and 72°C for 45 s. Due to the existence of GAPDH pseudogene and its amplification in DNA samples as well as cDNA, and to avoid its false positive results (will be explained in discussion), the PGM1 (Phosphoglucomutase1) a housekeeping gene was used as a control which could detect the presence of cDNA in the samples. PGM1 was amplified using PGM1-F (5'-TCCGACTGAGCGGCACT-GGGAGTGC-3')and PGM1-R (GCCCGCAGGTCC TCTT-TCCCTCACA-3') oligonucleotides with 30 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s. hTR was amplified using TR-F (5'-CGCCGTGCTTTTGCT CC-3') and TR-R (5'-ACTCGCTCCGTTCCTCTTCC-3') oligonucleotides for 5 cycles of 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, followed by 30 cycles of 94°C for 45 s, 63°C for 50 s, and 72°C for 45 s. Amplified products were subjected to electrophoresis in 2% agarose gels and were visualized with ethidium bromide.

Statistical analysis
The statistical analysis of the data was carried out by using the SPSS software package (SPSS Inc; Chicago, IL, USA; Version 11.5, 2003). The Pearson, Chi-square and Fisher Exact tests were used. The significance levels were considered for results with P value less than 0.05.
By using GAPDH primers in the control products (negative control groups 1 and 2), it was observed that 36 (72%) RNA extracts had DNA contamination. In total, 36 samples (with DNA contamination) demonstrated hTR expression in cDNA samples, whereas after DNase treatment, 17 samples did not show hTR expression.
The hTR expression in cDNAs was observed in 49 out of 50 (98%) samples, of which, 35 had telomerase activity. In one case without any DNA contamination and with telomerase activity, hTR expression in cDNA and DNased cDNA samples was not observed.
In cDNA samples with negative results for GAPDH-PCR, the hTR -PCR was found to be negative, which confirms the lack of any DNA contamination (Table 1).

Discussion
GAPDH has a psuedogene which could be amplified with GAPDH cDNA primers, as well as the current GAPDH gene cDNA. Usually, RNA is contaminated with DNA and due to the performance of PCR on a negative control group (this product has only probable DNA contamination), it will create a positive result that arises from the presence of a pseudogene in the sample (Fig. 2). The present subject was not considered in most studies in which the GAPDH housekeeping gene was used as a positive control to assess the accuracy of cDNA synthesis. In fact, the positive result of GAPDH-PCR may be due to the amplification of the pseudogene and not to the cDNA template. If GAPDH primers are used for DNA samples, then positive results will be occur. In this regard, this characteristic of GAPDH was used to ensure the accuracy of DNase treatment procedures.
When the result of GAPDH-PCR was positive in DNased treated cDNA samples, and negative in negative control group 2, then it can be assumed that the existing templates in DNased treated samples are cDNA and are not the result of any DNA contamination.
Furthermore, the effect of the probable remaining DNase activity post DNase enzyme inactivation was not noticed in previous studies using DNase treatment [27].
In this study, the accuracy of the procedures, including DNase treatment and cDNA synthesis and creating negative control groups were checked with GAPDH and PGM1 housekeeping genes. In the case of having a positive result for negative control groups, or a negative result for DNase treated cDNAs, then the repeat of the DNase treatment and use of negative control groups would be required.
A false estimation of our result, through a procedure without DNase treatment, would be expected, i.e., instead of observing 49 (98%) with hTR expression, the outcome could be decreased to 64%.  Electrophoregram for GAPDH and hTR PCR products