The Gene Expression of Cyclin D - Lab Report Example

Activity Semi-quantitative gene expression by reverse transcriptase- PCR (RT-PCR) analysis Objectives To study the gene expression of cyclin D1 inthe given breast tumour and normal tissue specimens at mRNA level using semi-quantitative reverse transcriptase PCR and agarose gel electrophoresis techniques. To determine the relative amount of cyclin D1 mRNA present in the given breast tumour and normal tissue specimens, and compare the amplified target cDNA to a constitutively expressed house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Introduction Dysregulated cell cycle control is a primary mechanism of cancer (Deshpande, Sicinski & Hinds, 2005). Alterations in the machinery that governs the progression from resting state into the cell cycle (the so-called G0 G1 transition) or from G1 into S phase are common to all tumor cells (Deshpande et al., 2005). It is the pRb pathway, which is the core decision-making machinery for progression from G1 to S phase that is commonly altered in several tumors. Metcalf (2010) described that cyclins are an evolutionarily conserved family of proteins that play an important role in the regulation of the cell cycle by binding to cyclin-dependent kinases (cdk). Normally, they are synthesized and destroyed in a precise manner and this periodicity enables specific cyclin-dependent kinase–cyclin complexes to facilitate the sequential events that take place during cell-cycle progression (Malumbres et al., 2009; Metcalf et al., 2010). The functions of cdk4 and cdk6, which are the partners of the D-type cyclins are dysregulated, which leads to an increased cell proliferation and turnover in mammalian cells (Metcalf et al., 2010). The D-type cyclins (D1, D2 and D3) are structurally and functionally similar proteins that are synthesized in response to mitogenic signals, bind to and form active complexes with cdk4 or cdk6, leading to phosphorylation and inactivation of pRb (Malumbres et al., 2009; Malumbres et al., 2004; Sherr, 1994; Metcalf et al., 2010). This in turn dissolves complexes of pRb with members of E2F family of transcription factors and associated chromatin-modifying enzymes, allowing transcription of genes required for S phase (Deshpande et al., 2005). According to a review by Deshpande et al. (2005), molecular analyses reveal the amplification, rearrangement of the cyclin D1 gene (localized on chromosome 11q13) as well as overexpression of cyclin D1 protein. It has been described in a wide spectrum of human cancers such as squamous cell carcinomas of head and neck, esophagus, tongue and larynx, carcinomas of uterine cervix, astrocytomas, non-small-cell lung cancers, soft-tissue sarcomas and others (Lammie et al., 1991; Reissmann et al., 1999; Rodrigo et al., 2000; Cheung et al., 2001; Fujii et al., 2001; Vielba et al., 2003). The review further comments that cyclin D1 is frequently involved in pathogenesis of human breast cancer (Deshpande et al., 2005). Thus, approximately 15–20% of human mammary carcinomas contain amplification of the cyclin D1 gene (Lammie et al., 1991; Buckley et al., 1993; Dickson et al., 1995), while cyclin D1 protein is overexpressed in over 50% of human breast cancers (Bartkova et al., 1994, 1995; Gillett et al., 1994; McIntosh et al., 1995; Gillett et al., 1996). Cyclins D2 and D3 genes are also amplified and their encoded proteins are overexpressed in many human cancers (Deshpande et al., 2005). Cyclin D2 is involved in B-cell lymphocytic leukemias and lymphoplasmacytic lymphomas (Delmer et al., 1995), chronic lymphocytic leukemias (Motokura & Arnold, 1993), as well as in testicular and ovarian germ cell tumors, while cyclin D3 overexpression in seen in glioblastomas, renal cell carcinomas, pancreatic adenocarcinomas and several B-cell malignancies such as diffuse large B-cell lymphomas or multiple myelomas (Buschges et al., 1999; Ito et al., 2001; Shaughnessy et al., 2001; Filipits et al., 2002; Hedberg et al., 2002). Similarly, overexpression of CDK4 is found (often as consequence of gene amplification) in breast cancers (An et al., 1999), gliomas, glioblastomas multiforme, sarcomas and meningiomas (Khatib et al., 1993; Schmidt et al., 1994; He et al., 1995; Wei et al., 1999; Simon et al., 2002). Reverse transcriptase polymerase chain reaction (RT-PCR) is a semi-quantitative technique and represents a powerful qualitative tool for detecting mRNA. Pfaffl (2004) in his comparison of real-time RT-PCR with reverse transcriptase PCR describes that, “During the plateau phase of the PCR there is no direct relation of "DNA input" to "amplified target"; hence the assay needs to be stopped at least in linear phase. The exponential range of amplification has to be determined for each transcript empirically by amplifying equivalent amounts of cDNA over various cycles of the PCR or by amplifying dilutions of cDNA over the same number of PCR cycles. Amplified RT-PCR end product is later detected by ethidium bromide (an insensitive stain) gel staining, radioactivity labelling, fluorescence labelling, high-performance liquid chromatography, southern blotting, densitometric analysis, or other post-amplification detection methods. This step-wise accumulation of post-PCR variability leads to semi-quantitative results with high intraassay and inter-assay variability”.(p. 91) The technique allows an mRNA molecule to be amplified as complementary DNA (cDNA) copies. In RT-PCR, the RNA strand is first reverse transcribed into cDNA using the enzyme reverse transcriptase and primers. Oligo(dT) is used as a universal primer for mRNA molecules because it binds to the endogenous poly(A) tail that all mRNA molecules posses. An RNA-dependent DNA polymerase or reverse transcriptase, obtainable from retroviruses, generates a cDNA to the target RNA molecule by extending the primer. The resulting cDNA is amplified by traditional PCR using gene-specific primers. The need to quantitate differences in mRNA expression and the availability of only small amounts of mRNA in some procedures such as in the use of: cells obtained by laser capture micro-dissection or small amounts of tissue or primary cells or precious reagents, led to the development of methods for the quantitation of mRNA (Hunt, 2010). These include real-time PCR (or reverse transcriptase real-time PCR), northern blotting, ribonuclease protection assays (RPA) and in situ hybridization. Northern blotting and RPAs are the gold standards, where no amplification is involved, but require more RNA whereas in situ hybridization is a qualitative technique (Hunt, 2010). As the spectrophotometric analysis cannot distinguish between non-degraded and degraded RNA, it is necessary to control for this additional source of variation by employing gene(s) whose expression remains constant (constitutively expressed) in the cells or tissues being investigated, following activation or proliferation of cells (Gause and Adamovicz, 1994). Therefore, gene expression data are often normalized to the expression levels of an internal control or so-called "housekeeping" genes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Other housekeeping genes include, beta actin mRNA, MHC I (major histocompatibility complex I) mRNA, Cyclophilin mRNA, mRNAs for certain ribosomal proteins e.g. RPLP0 (acidic ribosomal phosphoprotein P0, large, P0), 28S or 18S rRNAs (ribosomal RNAs), hypoxanthine-guanine phosphoribosyl transferase (HPRT) (Gause and Adamovicz, 1994; Hunt 2010). A housekeeping gene should be validated for the tissue being investigated. In order to quantitate the amount of cDNA, the amount of signal in the experimental sample is compared to an internal control for the target gene (Hunt 2010). Ratio target gene expression (experimental/control) = Fold change in target gene expression (expt/control) Fold change in reference gene expression (expt/control) RT-PCR indicates the levels of mRNA but does not give any evidence that the RNA is transcribed. However, the expression of cyclin D protein in cells can be detected by immunohistochemistry or western blot techniques. Method In the present experiment, a fixed amount of RNA (1µg) extracted from breast tumour and normal tissue specimens was reverse transcribed into cDNAs respectively with random primers. Commercial cDNA was used as a positive control. A relative reverse transcriptase PCR (RT-PCR) was performed using GAPDH as an internal standard to determine the mRNA relative expression levels for cyclin D1 in breast tumour and normal tissue specimens. The primer sequences for the specific target mRNA employed were: Cyclin D1 F: 5′-ACCCCGCACGATTTCATT-3′; Cyclin D1 R:5′-CACCTCCTCCTCCTCCTCTT-3′ ; GAPDH F: 5′- GTCAGTGGTGGACCTGACCT-3′; GAPDH R: 5′- AGGGGTCTACATGGCAACTG-3′ Each of the12 eppendorf tubes labeled respectively contained 3 µl of nuclease-free H2O and 5 µl 2X Master mix. The total volume of the PCR reaction mixture in each tube is 10 µl. 1µl cyclin D primers were added to tubes 1 to 6, and 1µl GAPDH primers were added to tubes 7 to 12 respectively. 1µl of tumour 1 cDNA sample was added to tubes 1 and 7 respectively. Similarly, 1µl of tumour 2 cDNA sample was added to tubes 3 and 9 respectively. 1µl of normal tissue 1 cDNA sample was added to tubes 2 and 8 respectively, and 1µl of normal tissue 2 cDNA samples was added to tubes 4 and 10 respectively. 1µl of positive control cDNA was added to tubes 5 and 11 respectively. The tubes labeled 6 and 12 contained 1µl nuclease-free H2O. PCR amplification for the specific genes was conducted for 35 cycles (94˚C for 1 min, 59˚C for 1 min and 72 ˚C for 1min) with a 10 min final incubation at 72˚C. The amplified products were subjected to 1.2% agarose gel electrophoresis and the bands were visualised under a UV transilluminator. Results Sharp clear bands were observed at approximately 750 bps and 550 bps which are the expected predicted sizes of the PCR products of GAPDH (36 kDa) and cyclin D (38 kDa) respectively. The lanes 1 to 5 showed a sharp band corresponding to cyclin D gene expression and the lanes 7 to 11 showed the presence of a band corresponding to.GAPDH gene expression. The results indicated the amplification of cyclin D and GAPDH in the positive control cDNA (lanes 5 and 11 respectively), breast tumour specimen-1 (lanes 1 and 7 respectively), breast tumour specimen-2 (lanes 3 and 9 respectively), normal tissue-1 (lanes 2 and 8 respectively), normal tissue-2 (lanes 4 and 10 respectively) [Gel picture 1 where, the MW ladder is on the extreme left] Activity 2: Extract total RNA from mammal tissues Objectives To extract total RNA from the given mammalian tissue sample and determine its concentration in the sample. Method The total RNA was isolated from the mammalian tissue sample using phenol, BCP (1-Bromo-3-chloroproane) and isopropanol. The extracted RNA sample was diluted in RNase-free water prior to spectrophotometry. Finally the concentration of the extracted RNA was determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. An absorbance of 1 unit at 260 nm corresponds to 40 μg of single-stranded RNA per ml. The volume of RNA sample obtained was 1.2 ml. Therefore, dilution = 4 μl of RNA sample + 996 μl distilled water (1/250 dilution). Measured absorbances of diluted sample in a 1 ml cuvette (RNase-free): A260 = 0.75, A280 = 0.384 Therefore, the concentration of RNA sample = 40 x A260 x dilution factor = 40 x 0.75 x 250 = 7500 μg/ml. Hence, the total yield = concentration x volume of sample in milliliters = 7500 μg/ml x 1.2 ml = 9000 μg = 9 mg RNA. “RNA samples must be free of contaminating proteins and other cellular material, organic solvents such as phenol or ethanol, and salts for performing gene expression analysis” (FAS Center for Systems Biology, 2008). By taking the ratio of a samples absorbance at 260nm to its absorbance at 280nm (A260/A280) the purity of the sample was determined. A very pure sample of RNA with minimal protein and degradation will have A260/A280 between 1.8 and 2 (FAS Center for Systems Biology, 2008). The A260/A280 of the total RNA in the present experiment was 1.953, which indicates a pure RNA sample with no degradation. Isolation of intact RNA is essential for many techniques used in gene expression analysis. Hence, it is necessary to check RNA integrity regardless of the downstream application. The integrity of RNA could be determined by running an aliquot of the RNA preparation on 1% denaturing agarose gel stained with ethidium bromide (EtBr) (FAS Center for Systems Biology, 2008). A high-quality preparation of mammalian total RNA is characterized by two sharp, clear bands at approximately 4.5 and 1.9 kb, representing 28S and 18S ribosomal RNA, and the absence of genomic DNA (FAS Center for Systems Biology, 2008). Ambions TechNotes (2001) in one of its articles discusses the quality and methods to check the integrity of RNA. “The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a very low molecular weight smear. Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly”. “A simple test for genomic DNA contamination is to employ the obtained RNA directly as a template in a PCR reaction with primers for any well characterized gene, such as beta-actin or GAPDH” (FAS Center for Systems Biology, 2008). The designed primers are chosen in such a way that they do not span a large intron and amplify a short fragment ( Read More