Preparation of LZT-Hs8 genes; a family member to the LIV-1subfamily of zinc transporter, for further cloning strategy.
Materials and Methods
2.0.1 Chemical and Reagents
BD Vacutainer 4.0 ml Sodium Heparin(NH) tube. SYSTERM chemAR products such as chloroform, isopropanol and ethanol. GIBCO RNAse Free. Molecular BioProduct RNase away. Vivantis Agarose (molecular Biology Grade) .BIO BASIC INC Ethidium Bromide (EtBr) Staining Solution.Easy-REDTM Total RNA Extraction Kit, 100 ml is purchased from iNtRON BIOTECHNOLOGY.
Fermentas products such as, 10X TBE electrophoresis buffer, GeneRulerTM 1 kb DNA Ladder, ready- to- use, 1µg total RNA, 10µg random hexamer primer, 0.156mM dNTP mixture, 0.01M dithiothreitol (dTT),1 x PCR buffer B, M-MuLV Reverse Transcriptase, DNase I, RiboLockTM RNase Inhibitor, 0.5µM of 3' and 5' primers of LZT-Hs8, 0.25mM of dNTP's mix, 1 X PCR buffer, nuclease free water and 6X DNA loading dye and KAPABIOSYSTEMS product KAPATaq DNA polymerase are purchased from XZIZ BIOTECH SDN BHD.
Easy-REDTM is selected to process large quantity of liquid samples such as serum for RNA isolation. The bottle is folded with aluminum foil and must keep in the dark at all time. To prepare 100ml of 70% Ethanol, 70ml of 95% ethyl alcohol measured and 30ml of distilled water added. To prepare 1X TBE buffer, 10ml of 10X TBE buffer added to 90ml of distilled water and mixed well.
ESCO BIOTECH Laminar flow cabinet to provide sterilized working environment. Purposely used during RNA extraction to avoid breathing vapor of easy-REDTM . Gel electrophoresis apparatus purchased from MAJOR SCIENCE innovative life science tools.Pacific Image Electronics Co.,Ltd Ultraviolet transilluminator and Mbiotech digibox7000. Centrifuge 5418 and dualfilter 10 µl PCR purchased from eppendorf. Refrigerators such as -40C SHARP Deodorizer, -200C ACSON international and -800C Revco Technologies.
Disposable plastic utilities ; dualfilter appendorf TIPS 10 µl pipette tips, AXYGENE scientific 100 µl pipette tips , AvantGuard Barrier tips,1000 µl pipette tips.1.5 ml and 0.2 ml microcentrifuge tubes are purchased from medigene Sdn Bhd and chemopharm Sdn Bhd.PECHINEY PLASTIC PACKAGING parafilm “M” laboratory film.
2.0.3. Autoclave procedure for material sterilization
For sterilization purpose, all the disposable tubes and tips were autoclaved. First, materials to be autoclave are placed in autoclavable bag or container, and then autoclave indicator tape placed on each material's bags or container to check after the run is completed, this tape has changed color from yellow to black stripes. Second, autoclave machine is filled with100ml of distilled water and the materials are placed into the machine using metal filter-pan. Finally, time is set for 20 minutes and exposure temperature at 121 0 C to start the process of autoclaving.
2.1.1. Blood sample
Firstly, blood sample of Malaysian female breast cancer patients who have undergone treatment were collected at University Malaya Medical Centre, UMMC. Secondly, permission was obtained from patients to withdraw blood in agreement with etiquettes approved by UMMC. Thirdly, consent form attached with the general information about this project was distributed to the patients to read and was explained to them before collection procedure. Fourthly, medical record of every patient gathered to make comparison on each sample.
Subsequently, in Clinical Investigation Centre (CIC) of UMMC, collected blood samples were spin down without delay to separate into cold blood and serum and the upper layer which is serum taken out cautiously without disturbing the bottom layer and transferred to microcentrifuge tubes. Finally, tubes were stored at -800C refrigerator for storage.
2.1.2. RNA extraction
RNA extraction is performed to the purify RNA from patient serum samples. First of all, to prepare RNA 750 µl of easy-REDTM solution added to 250 µl of sample which is prepared in 1.5 ml microcentrifuge tube and placed on ice immediately. Easy-REDTM solution is added for the purpose of sustaining the quality and integrity of RNA and to promote protein denaturation. Secondly, pipette is used to pass the undissolved mixture for some time, so as to lysis the cell in the sample. The volume ration of easy-REDTM solution to sample always ensured to 3:1. Thirdly, the sample is mixed by vortex rapidly in room temperature for 15 sec and kept warm for 5 min time in room temperature.
Fourthly, once there is no cluster seen in the sample after vortex 200 µl of Chloroform added and mixed with sample by vortexes rapidly in room temperature for 15 sec and kept warm for 5 min time in room temperature. Phenol layer is separated from aqueous layer and finally to isolate DNA and RNA or genomic protein with the action of chloroform.
Fifthly, tubes are centrifuged at 13,000 rpm (40C) for 15 min time. Once done with centrifugation the 400 µl of upper fluid is transferred to a new 1.5 ml centrifuge tube. This is the first centrifugation where two layers formed in the solution observed. First layer is consisting of RNA whereas the second layer is phenol layer that is red color produced which is consisting of protein or cell debris and others. There is a formation of white sediments at the edge in between the two layers observed which contains combination of protein and genomic DNA.
Subsequently, 400 µl of isopropanol is added so as to equivalent volume with the solution after first centrifugation and tube is inverted for 5 times to finely mix up with it. Formation of white layer that contains RNA observed.
Then, second centrifugation at 13,000 rpm (40C) for 10 min time is carried out and supernatant is removed cautiously without touching the pellet at the bottom of the tube.
Following this, 1 ml or 1000 µl of 70% ethanol is added and inverted for 5 times to finely mix up with the solution. Then, proceeded with the final centrifugation at 13,000 rpm (40C) for 10 min time to wash away the impurities like salt and supernatant is taken out without touching the white RNA pellet. Consequently, the RNA pellet is changed to white due to dehydration after the centrifugation of mixture observed. At that moment, the left over RNA left to dry and uncapped and did not leave to dry extremely in order to maintain its solubility.
Finally, RNA is dissolved in 30 µl of RNase free water, where mixture is passed for some times by using a pipette tip. Then, resulted RNA is stored at -800C to prevent RNA degradation.
RT-PCR is a technique performed to transcript mRNA into its complementary DNA (cDNA) by the reaction of enzyme reverse transcriptase and the produced cDNA is amplified using PCR to generate many copies.
First of all, 11µl of RT-PCR mixture solution is prepared in the 0.2ml microcentrifuge tube; components are 3µl of total RNA, 1.0 µl of random hexamer primer, 1.0 µl of dNTP mixture, 1.0 µl of dithiothreitol (dTT) and 5.0 µl of 1 x PCR buffer B.
Secondly, mixture solution is denatured at 950C for 5 minutes in the PCR thermal cycler. Thirdly, once done with denaturation the mixture solution is taken out from the PCR thermal cycler and placed on the ice for 5 minutes immediately. Fourthly, mixture solution was spin down for 1 minute at 13,000 rpm in centrifuge.
Fifthly, mixture solution is further added with 0.5 µl of RNase inhibitor and 1 µl MMLV reverse transcriptase on ice to prevent from RNA degradation. Following this, mixture solution is incubated for 40 minutes at 420C in the PCR thermal cycler. Subsequently, temperature is increased up to 950C for 5 minutes for denaturation. Finally, the resulted cDNA are stored in -200C for later use for PCR.
PCR is a technique performed to amplify DNA from a tiny sample to produce many copies of identical DNA in a short period of time.
First of all, PCR master mix is prepared; consist of 0.6 µl of dNTP's mix, 0.12 µl of 3' and 5' primers of LZT-Hs8, 2.4 µl of 1 X PCR buffer, 19.7 µl nuclease free water, and 0.08 µl of KAPATaq DNA polymerase.
Secondly, master mix is mixed gently by pipetting up and down for some time and 1.0 µl of template DNA is added to the each tube containing the master mix.
Thirdly, The PCR tubes are placed in the thermal cycler and cycling program is started by following the order of processes ; denaturation of template at 95°C for 1 minute and followed by, annealing of primers at 570C for 1 minute. These programs were repeated for 31 cycles .Subsequently, elongation of product at 72°C for 1 minute performed. Finally, termination at 72°C for 10 minutes is performed to obtain better exposure on agarose gels. After amplification, samples are stored at -20°C for longer term storage.
2.1.5. Gel electrophoresis
Gel electrophoresis is a technique which is carries out for analytical purposes, usually after amplification of DNA through PCR for separating DNA fragments by size. First of all, to prepare gel 1% of agarose powder added and 1X TBE solution into a glass flask. Secondly, the opening and neck of glass flask is covered with plastic wrap and solution mixed by swirling before microwave the agarose solution at high heat until boiling. Solution heated until all crystals are completely dissolved and leave to cool before pouring into the tray. Thirdly, 1µl of 10mg/ml Ethidium Bromide (EtBr) added into the solution. Fourthly, agarose solution poured in the tray and any bubbles that do happen can be free from unevenness of surface by isolating using a pipette tip to the edge of the gel. Fifthly, comb placed on the edge of gel surface to create wells and leave to solidify for 30 minutes.
Once the gel became solid, comb is removed and gel along with the tray transferred to the electrophoresis tank .subsequently, electrophoresis tank is filled with buffer until the gel is flooded with buffer. DNA Samples, loading buffer and DNA ladder are prearranged on parafilm before loaded into the wells; 10 µl of DNA ladder, 3µl of Loading dye and 3µl of DNA Sample.
Following this, DNA ladder and DNA samples which is mixed with loading dye are loaded into the wells, to load samples pipette tip inserted deep into the well and pushed out the liquid slowly. Electrodes are connected so that the DNA migrated towards the anode or positive electrode from negative electrode. Gel is allowed to run at 75 Volts and for 30 minutes. Finally, gel is placed on Ultraviolet transilluminator to view and photographed as a result.