1 Effect of Maternal Estradiol Hormone on Gene Expression in Zebrafish (Danio rerio) Embryos and its Clinical Implications Md. Golam Rabbane 1 and A. F. M. Arifur Rahman 2 1 Department of Fisheries, University of Dhaka, Dhaka-1000, Bangladesh 2 Department of Fisheries and Marine Science, Noakhali Science and Technology University, Sonapur, Noakhali, Bangladesh ABSTRACT: Maternal steroid hormone, estradiol plays significant role in fertilized eggs by performing proper embryo development. The study was performed to observe the genetic effects of estradiol by using reverse transcriptase polymerase chain reaction. A total three steroid responsive genes were analysed namely anx2a, ccng1 and hmox1. The ef1a gene was used as reference to normalize data. The transcripts of anx2a were down regulated in E treated embryos at 8 hpf when compared to contol and E2 0.1 treated embryos. The ccng1 transcripts for E2 0.1 and E and E2 treated embryos were equally and strongly expressed. There was also no up or downregulation when analysed with hmox1 gene. So the results suggest that different estradiol concentrations can alter specific genetic expression at 8 hpf embryonic state and clinically it could be used as an effective gene therapy. However, extensive studies should be conducted to assess its beneficial effects on human being. Key words: Gene expression, Estradiol hormone, Zebrafish, Clinical implications INTRODUCTION In teleost fish like other oviparous vertebrates, the yolk of ovulated eggs contain significant amount of liposoluble hormones such as thyroid, steroid and retinoid etc. These hormones are taken up from either maternal circulation or follicular envelopes. 1,2 This store of maternal hormones may accomplish the regulatory requirements of larvae for growth, development, osmoregulation, stress responses and other physiological functions prior to the functional development of their own endocrine glands. 3 Experiments on some fish species have shown that hormones especially maternal steroid hormones play important role in larval development and thus may affect egg quality. 4 For example, several sex steroids have been reported to be present in fertilized eggs of tilapia and salmonids, cortisol in Mozambique tilapia (Oreochromis mossambicus) and Correspondence to: Md. Golam Rabbane Cell: Dhaka Univ. J. Pharm. Sci. 12(2): , 2013 (December) testosterone in medaka (Oryzias latipes). 5-8 In addition, dynamic changes in cortisol content profile were reported in tilapia and zebrafish, showing a steady decline from fertilization until hatching in both species. 9,10 Estradiol or 17β-estradiol is an important sex hormone. Estradiol is abbreviated E2 as it has two hydroxyl groups in its molecular structure. E2 has not only a critical impact on reproductive and sexual functioning, but also affects other organs, including the bones. So the studies was undertaken to investigate the genetic effects of estradiol on fertilized zebrafish eggs. MATERIALS AND METHODS Breeding and treatment. Natural breeding was performed to obtain fertilized eggs with 28.5 o C water temperature and with a photoperiod of 14 h light and 10 h dark. Immediately after fertilization, fertilized eggs were pooled in estradiol (0.1 mg/l and 0.01 mg/l), and abs EtOH (1 mg/l) solutions for 2 h for development and fixation up to 8 hours post
2 148 Rabbane and Rahman fertilization (hpf). After 2 h of treatment, the estradiol and EtOH treatment solution was poured out and eggs were gently washed 5-fold in fish water to eliminate any trace of estradiol and ethanol. RNA isolation. To evaluate gene expression, total RNA was extracted from pools of about 60 embryos at 8 hpf developmental stages using TRIZOL reagent, according to the manufacturer s instructions (Invitrogen, Milan, Italy). The experimental phase of extraction and manipulation was carried out under a chemical hood using sterile glassware or sterilized at 200 C in oven for the entire night. The isolated RNA samples were stored at -80 C until future use. RNA and cdna samples, obtained from embryos and PCR (Polymerase Chain Reaction) reactions were analysed by agarose (Fisher Molecular Biology, USA) gel electrophoresis. This was carried out until the marker dye (bromophenol blue, added to the sample prior to loading) reaches the end of the gel. The nucleic acids in the gel are visualised by staining with the intercalating dye gel red and examined under ultraviolet light. Quantification of total RNA. The concentration of total RNA was measured by NanoDrop Spectrophotometer (Celbio, Milan, Italy), which allows an assessment of the state of purity or protein contamination. The concentration of 1.5 µl solution of nucleic acid was determined by measuring the absorbance at 260 nm. An A 260 of 1.0 is equivalent to a concentration of 50 µg/ml for double-stranded DNA, or 40 µg/ml for single-stranded DNA or RNA. The A 260 /A 280 ratio should be 1.8 for pure DNA and 2.0 for pure RNA preparations. 11 Reverse transcription of RNA. After quantification of extracted RNA, reverse transcription was performed using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) protocol (Invitrogen), according to the manufacturer s instructions. M-MLV RT uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand. The M-MLV RT PCR is a two-step process. Briefly, 2 µg of the purified total RNA was mixed with random hexamer and pure water to a final volume of 10 µl, denatured by incubation at 70 o C for 5 min and then placed on ice for 5 min. Then, 15 µl of cdna synthesis Buffer (5X), dntp Mix (10 mm), M-MLV reverse transcriptase enzyme and H 2 O were added to each sample. The samples were incubated in a PCR machine at 25 C for 10 min, 50 C for 50 min and 70 C for 15 min. Then, the cdna reactions were either stored at -20ºC or used for PCR immediately. Touchdown PCR. Total cdna (200 ng) was amplified by PCR containing 4µl of ImProm-II 5X Reaction Buffer, 2.4 µl of MgCl 2, 1 µl of dntp mix, 0.5 µl of Recombinant RNasin Ribonuclease Inhibitor, 0.5 µl of the respective primers, 1 µl ImProm-II Reverse Transcriptase and ultrapure water to a final volume of 10 µl. The melting point of the primer sets the upper limit on annealing temperature. The annealing temperature of the initial cycle is 5-10 C above the melting temperature of the primers and the temperature is gradually reduced by 1 C for each following cycle down to the lowest melting temperature value of the primer. PCR products for each gene of interest were sequenced to confirm amplicon identity. The list of primers are shown in Table 1. Table 1. List of primers Primer Orientation Sequence Position Accession number ef1a-f Sense 5 -GACAAGAGAACCATCGAG ef1a-r Antisense 5 -CCTCAAACTCACCGACAC ccng1-f Sense 5 -GATTGAGGATCAGCACGAG ccng1-r Antisense 5 -CAGTTATGGGCACTCAACAC anx2a-f Sense 5 -GCACAGATGTGAAGTGCTG anx2a-r Antisense 5 -CAGTCGTCTCCATTGCAC hmox1-f Sense 5 - CCACACACCGATATGCAC hmox1-r Antisense 5 -CAACGTGATGCCCACTCC NM_ NM_ NM_ NM_
3 Effect of Maternal Estradiol Hormone on Gene Expression 149 RESULTS AND DISCUSSIONS The quality of RNA was identified by running the extracted RNA samples through agarose gel electrophoresis by indicating two bands: an upper 28S rrna and a lower 18S rrna (Figure 1). So the quality of extracted RNA was found good due to the absence of additional bands or smear. Figure 1. Separation of total RNA on denaturing gel electrophoresis followed by gel red staining. The quantification results of extracted RNA is shown in table 2 where E and E2 0.1 received higher concentration and ng/µl respectively. But lower concentration observed ng/µl in E2 control sample. Samples E and E2 control obtained pure quality RNA because A 260 /A 280 was greater than To determine the effects of estradiol on gene expression during first developmental stage, a total three genes were selected namely annexin A2a (anx2a), cyclin G1 (ccng1) and heme oxygenase (decycling)1 (hmox1). The touchdown PCR analysis revealed that the transcripts of anx2a were poorly expressed in E treated embryos when compared to control and E2 0.1 treated embryos as shown in Figure 2. But there was no anx2a transcripts different between E2 0.1 and control embryos. It may be assumed that biological mechanisms can influence the regulation of anx2a transcripts with changing the amount of E2. This messenger was up-regulated when embryos were knocked down with MO2-esr2a. 12 anx2a plays vital role as calcium ion binding, calcium-dependent phospholipid binding and cytoskeletal protein binding So these binding capacities could be reduced by down-regulating anx2a gene expression. ccng1 transcripts for all treatments were strongly and equally expressed at 8 hpf stage embryos (Figure 3). The transcript of cyclin G1 (ccng1), that encodes a protein with intrinsic growth inhibitory activity, was found to be up-regulated at 48 hpf when knocked down with MO2-esr2a. 12,15 Table 2. Quantification of extracted RNA by NanoDrop Spectrophotometer. Sample ID ng/µl A 260 A 260 /A 280 E E E2 cont Figure 2. Expression of anx2a transcripts during 8 hpf Figure 3. Expression of ccng1 transcripts during 8 hpf Finally, the expression of heme oxygenase (decycling)1 (hmox1) transcripts were analysed to determine the effects of estradiol. Hmox1 encodes a protein that in mammals, catabolizes heme to biliverdin, carbon monoxide, and free iron and is involved in iron homeostasis. 16 In zebrafish, this gene is expressed in the extraembryonic yolk syncytial layer, lens, and a small population of blood cells. 17 There was no up or down-regulation when
4 150 Rabbane and Rahman analysed with hmox1 gene (Figure 4). Significant down-regulation of hmox1 transcripts by RT-PCR at 8 and 48 hpf developmental stages when knocked down with MO2-esr2a. 12 Estradiol has been attached to the expansion and progression of cancers such as ovarian cancer and endometrial cancer. Estradiol effects target tissues by interacting with estrogen receptors. These estrogen receptors are involved in gene expression. 18 When hormone binds to the estrogen receptors, it possibly causes damage to the DNA, increase in cell proliferation and DNA replication. 19 Figure 4. Expression of hmox1 transcripts during 8 hpf The present report reveals that oocyte estradiol concentration can change genetic expression at different developmental stages in zebrafish. So a powerful regulatory control linked to maternal experience in terms of estradiol can be directly transmitted into lineage before their actual sensing of surrounding environmental cues. In this way estradiol transcripts in oocyte may expect an epigenetic plasticity in zebrafish development, as a maternal contribution to the adaptive value of the progeny. ACKNOWLEDGEMENT The authors would like to thank Prof. L. Colombo, Dr. L.D. Valle and Dr. F. Benato for their kind assistance to conduct this study. REFERENCES 1. McCormick, M.I Experimental test of the effect of maternal hormones on larval quality of a coral reef fish. Oecologia 118, Irie, T. and Seki, T Retinoid composition and retinal localization in the eggs of teleost fishes. Comp. Biochem. Physiol. Biochem. Mol. Biol. 131, Lam, T.J Hormones and egg larval quality in fish. J. World Aquaculture Society. 25, Brooks, S., Tyler, C.R. and Sumpter, J.P Egg quality in fish: what makes a good egg? Rev. Fish Biol. Fish. 7, Rothbard, S., Moav, B. and Yaron, Z Changes in steroid concentrations during sexual ontogenesis in tilapia. Aquaculture 61, Feist, G., Schreck, C.B., Fitzpatrick, M.S. and Redding, J.M Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. Gen. Comp. Endocrinol. 80, Shiraishi, K., Matsuda, M., Mori, T. and Hirano, T Changes in expression of prolactin- and cortisol-receptor genes during early-life stages of euryhaline tilapia (Oreochromis mossambicus) in fresh water and seawater. Zool. Sci. 16, Iwamatsu, T., Kobayashi, H., Sagegami, R. and Shuo, T Testosterone content of developing eggs and sex reversal in the medaka (Oryzias latipes). Gen. Comp. Endocrinol. 145, Hwang, P.P., Wu, S.M., Lin, J.H. and Wu, L.S Cortisol content of eggs and larvae of teleosts. Gen. Comp. Endocrinol. 86, Aslop, D. and Vijayan, M.M Development of the corticosteroid stress axis and receptor expression in zebrafish. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R711-R Nicholl, D.S.T An introduction to genetic engineering. Cambridge University Press, p Celeghin, A., Benato, F., Pikulkaew, P., Rabbane, M.G., Colombo, L. and Dalla Valle, L The knockdown of the maternal estrogen receptor 2a (esr2a) mrna affects embryo transcript contents and larval development in zebrafish. Gen. Comp. Endocrinol. 172, Singh, S.K., Sundaram, C.S., Shanbhag, S. and Idris, M.M Proteomic profile of zebrafish brain based on twodimensional gel electrophoresis matrix-assisted laser desorption/ionization ms/ms analysis. Zebrafish 7, Xiong, X.P., Dong, C.F., Xu, X., Weng, S.P., Liu, Z.Y. and He, J.G Proteomic analysis of zebrafish (Danio rerio) infected with infectious spleen and kidney necrosis virus. Dev. Comp. Immunol. 35, Zhao, L., Samuels, T., Winckler, S., Korgaonkar, C., Tompkins, V., Horne, M.C. and Quelle, D.E Cyclin G1 has growth inhibitory activity linked to the ARF-Mdm2- p53 and prb tumor suppressor pathways. Mol. Cancer Res. 1,
5 Effect of Maternal Estradiol Hormone on Gene Expression Poss, K.D. and Tonegawa, S Heme oxygenase 1 is required for mammalian iron reutilization. Proc. Natl. Acad. Sci. 94, Thisse, B. and Thisse, C Fast Release Clones: A High Throughput Expression Analysis. ZFIN Direct Data Submission. (http://zfin.org). 18. Sreeja, S. Kumar, T. Lakshmi, B. and Sreeja, S Pomegranate extract demonstrate a selective estrogen receptor modulator profile in human tumor cell lines and in vivo models of estrogen deprivation. J. Nutr. Biochem. 23, Christoforos, T. Strom, A. Lindberg, K. and Gustafsson, J Estrogen receptor beta decreases survival of p53- defective cancer cells after DNA damage by impairing G2/M checkpoint signaling. Breast Cancer Res. Treat. 127,
HiPer RT-PCR Teaching Kit Product Code: HTBM024 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 4 hours Agarose Gel Electrophoresis: 45 minutes Storage Instructions: The
Lecture 18 PCR Technology Growing PCR Industry Basic PCR, Cloning of PCR product, RT-PCR, RACE, Quantitative PCR, Multiplex PCR, Hot start PCR, Touchdown PCR,PCR sequencing.. How PCR started The DNA duplex
DNA AMPLIFICATION & PCR AMV First Strand cdna Synthesis Kit Instruction Manual NEB #E6550S Store at 20 C ISO 9001 Registered Quality Management ISO 14001 Registered Environmental Management ISO 13485 Registered
DNA AMPLIFICATION & PCR ProtoScript First Strand cdna Synthesis Kit Instruction Manual NEB #E6300S/L 30/150 reactions Version 2.2 11/16 be INSPIRED drive DISCOVERY stay GENUINE This product is intended
PHENIX PCR Enzyme Guide PHENIX offers a broad line of premium quality PCR Enzymes. This PCR Enzyme Guide will help simplify your polymerase selection process. Each DNA Polymerase has different characteristics
DNA AMPLIFICATION & PCR ProtoScript II First Strand cdna Synthesis Kit Instruction Manual NEB #E6560S/L 30/150 reactions Version 1.5 12/17 be INSPIRED drive DISCOVERY stay GENUINE This product is intended
Cat. # 6110A For Research Use PrimeScript 1st strand cdna Synthesis Kit Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Materials Required but not Provided... 3 IV. Storage...
Reverse transcription-pcr (rt-pcr) Dr. Hani Alhadrami email@example.com www.hanialhadrami.kau.edu.sa Overview Several techniques are available to detect and analyse RNA. Examples of these techniques
Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single
T7-Based RNA Amplification Protocol (in progress) Jacqueline Ann Lopez (modifications) Amy Cash & Justen Andrews INTRODUCTION T7 RNA Amplification, a technique originally developed in the laboratory of
Genetic Engineering & Recombinant DNA Chapter 10 Copyright The McGraw-Hill Companies, Inc) Permission required for reproduction or display. Applications of Genetic Engineering Basic science vs. Applied
Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously
Cat. # RR086A For Research Use One Step SYBR PrimeScript RT-PCR Kit II Product Manual Table of Contents I. Description...3 II. III. IV. Principle...3 Components...5 Storage...6 V. Features...6 VI. VII.
Methods of Biomaterials Testing Lesson 3-5 Biochemical Methods - Molecular Biology - Chromosomes in the Cell Nucleus DNA in the Chromosome Deoxyribonucleic Acid (DNA) DNA has double-helix structure The
Cat. # RR036A For Research Use PrimeScript RT Master Mix (Perfect Real Time) Product Manual Table of Contents I. Description... 3 II. Kit Components... 3 III. Materials Required but not Provided... 3 IV.
Viral Detection Animal Inoculation Culturing the Virus Definitive Length of time Serology Detecting antibodies to the infectious agent Detecting Viral Proteins Western Blot ELISA Detecting the Viral Genome
APPLICATION NOTE SuperScript IV Reliable and sensitive RT-qPCR analysis of whole-blood RNA samples Introduction Most commercially available reverse transcription products do not perform well with difficult
Product Name : Simple mirna Detection Kit Code No. : DS700 This product is for research use only Kit Contents This kit provides sufficient reagents to perform 20 reactions for detecting microrna. Components
QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 007) Please contact Center for Plant Genomics (CPG) facility manager Hailing Jin (firstname.lastname@example.org) regarding questions or corrections.
Score winning cdna yields with RT SuperScript III offers: Higher cdna yields Higher thermal stability Longer half-life than any other RT you could use Announcing SuperScript III Reverse Transcriptase How
Molecular Cloning Methods Mohammad Keramatipour MD, PhD email@example.com Outline DNA recombinant technology DNA cloning co Cell based PCR PCR-based Some application of DNA cloning Genomic libraries
2054, Chap. 14, page 1 I. Recombinant DNA technology (Chapter 14) A. recombinant DNA technology = collection of methods used to perform genetic engineering 1. genetic engineering = deliberate modification
XactEdit Cas9 Nuclease with NLS User Manual An RNA-guided recombinant endonuclease for efficient targeted DNA cleavage Catalog Numbers CE1000-50K, CE1000-50, CE1000-250, CE1001-250, CE1001-1000 Table of
PureSpin DNA Clean Up Kit Micro Columns INSTRUCTION MANUAL KIT COMPONENTS For Research Use Only PureSpin DNA Clean Up Kit, Micro Columns w/out Caps (Kit Size) OD2080 (50 Preps.) OD2080-2 (200 Preps.) Storage
USB HotStart-IT for increased specificity and consistent results PCR, qpcr and qrt-pcr USB PCR Reagents Choose USB HotStart-IT products for increased specificity and consistent results. Long and Accurate
Supporting Information Wiley-VCH 2006 69451 Weinheim, Germany Rolling-circle Amplification of a DNA Nanojunction Chenxiang Lin, Mingyi Xie, Julian J.L. Chen, Yan Liu and Hao Yan A. RCA replication of the
1 Laboratory #7 Polymerase chain reaction () is DNA replication in a test tube. In vitro enzymatic amplification of a specific segment of DNA. Many Applications. direct cloning from DNA or cdna. Mutagenesis
Low cost and non-toxic genomic DNA extraction for use in molecular marker studies. Version 1.4, February 28 th, 2013. Prepared by Bernhard Hofinger, Owen Huynh and Brad Till. 1. OBJECTIVE To develop and
Cat. # RR037A For Research Use PrimeScript RT reagent Kit (Perfect Real Time) Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Storage... 3 IV. Features... 4 V. Precautions...
Second Edition December October 2010 2005 QIAGEN LongRange 2Step RT-PCR Handbook For reliable and accurate long-range two-step RT-PCR up to 12.5 kb Sample & Assay Technologies QIAGEN Sample and Assay Technologies
History of recombinant DNA technology Recombinant DNA Technology (DNA cloning) Majid Mojarrad Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists
mmu-mir-200a-3p Real-time RT-PCR Detection and U6 Calibration Kit User Manual Catalog # MBS826230 For the detection and quantification of mirnas mmu-mir-200a-3p normalized by U6 snrna using Real-time RT-PCR
2x PCR LongNova-RED Components RP85L 100 reactions (50 μl) RP85L-10 1000 reactions (50 μl) 2x PCR LongNova-RED 2 x 1.25 ml 20 x 1.25 ml PCR grade water 2 x 1.5 ml 20 x 1.5 ml Storage & Shiing Storage conditions
sirna Transfection Into Primary Neurons Using Fuse-It-siRNA This Application Note describes a protocol for sirna transfection into sensitive, primary cortical neurons using Fuse-It-siRNA. This innovative
Section A: DNA Cloning 1. DNA technology makes it possible to clone genes for basic research and commercial applications: an overview 2. Restriction enzymes are used to make recombinant DNA 3. Genes can
The Polymerase Chain Reaction Chapter 6: Background Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off the road and outlined a way to conduct DNA replication in a tube Worked for
PRODUCT INFORMATION Long PCR Enzyme Mix #K0182 500 u Lot Exp. 00.0000 Store at -20 C. CERTIFICATE OF ANALYSIS Long PCR Enzyme Mix is functionally tested in PCR amplification of 47.4 kb fragment from lambda
Laboratory for Environmental Pathogens Research Department of Environmental Sciences University of Toledo Polymerase Chain Reaction (PCR) Background information The polymerase chain reaction (PCR) is an
Proteomics And Cancer Biomarker Discovery Dr. Zahid Khan Institute of chemical Sciences (ICS) University of Peshawar Overview Proteomics Cancer Aims Tools Data Base search Challenges Summary 1 Overview
Cloning Small RNAs for Sequencing with 454 Technology Protocol provided by Dr. Greg Hannon, Cold Spring Harbor Laboratory 1. RNA preparation 1. Total RNA is isolated from tissue or cells with TRIZOL followed
Instructions for Use Life Science Kits & Assays Content Content 1 Product and order number... I 2 Storage conditions... I 3 Description... II 3.1 Quality data... II 3.2 Unit definition... II 4 Delivered
HiPer Random Amplification of Polymorphic DNA (RAPD) Teaching Kit Product Code: HTBM031 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 3.5 hours Agarose Gel Electrophoresis:
mmu-mir-34a Real-time RT-PCR Detection Kit User Manual Catalog # CPK1272 For the detection and quantification of mirna mmu-mir-34a using Real-Time RT-PCR detection instruments. For research use only. Not
Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms No. 1 of 10 1. The mouse gene knockout is based on. (A) Homologous recombination (B) Site-specific recombination
AP Biology Gene Expression/Biotechnology REVIEW Multiple Choice Identify the choice that best completes the statement or answers the question. 1. Gene expression can be a. regulated before transcription.
HiPer Gel Extraction Teaching Kit (Column Based) Product Code: HTBM010 Number of experiments that can be performed: 10 Duration of Experiment Agarose Gel Electrophoresis: 1 hour Protocol: 1 hour Agarose
De gekoppelde afbeelding kan niet worden weergegeven. Het bestand is mogelijk verplaatst, heeft een andere naam gekregen of is verwijderd. Controleer of de koppeling verwijst naar het juiste bestand en
QuickClean II Gel Extraction Kit Cat. No. L00418 Technical Manual No. TM0594 Version: 03042011 I II III IV V VI VII VIII Description......1 Components.....1 Storage.... 1 Technical Information....1 Protocol.....2
User Manual Mir-X mirna First-Strand Synthesis and SYBR qrt-pcr User Manual United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Clontech Laboratories,
Amino-allyl Dye Coupling Protocol Joseph DeRisi, June 2001 Typically, fluorescently labeled cdna is generated by incorporation of dyeconjugated nucleotide analogs during the reverse transcription process.
1 Multiple choice questions (numbers in brackets indicate the number of correct answers) February 1, 2013 1. Ribose is found in Nucleic acids Proteins Lipids RNA DNA (2) 2. Most RNA in cells is transfer
HiPer Real-Time PCR Teaching Kit Product Code: HTBM032 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1.5 hours Storage Instructions: The kit is stable for 12 months from
March 2011 RT 2 Easy First Strand Handbook For cdna synthesis Sample & Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies,
mirna qrt-pcr Detection Kit For quantitative detection of mature mirna Cat. No. QP015 (20 RT and 200 qpcr reactions) Cat. No. QP016 (60 RT and 600 qpcr reactions) Used in combination with the All-in-One
Principals of Real-Time PCR Amira A. T. AL-Hosary Lecturer of Infectious Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt What Is Real-Time PCR? Nucleic acid (DNA) amplification and detection
Molekulargenetische Reagenzien Raumtemperaturstabile PCR und qpcr Reagenzien Polypeptide Stabilization Technology: Stability TAG Ice-free reaction set-up Our temperature stable enzymes allow you to change
Guidelines for Preventing Contamination of PCR During PCR more than 10 million copies of a template DNA are generated. Therefore, care must be taken to avoid contamination with other templates and amplicons
Manual 15 Reactions LEXSY in vitro Translation Cell-free protein expression kit based on Leishmania tarentolae for PCR-based template generation Cat. No. EGE-2010-15 FOR RESEARCH USE ONLY. NOT INTENDED
RNA Isolation and Technology Applications Nadine Nassif Senior Research Scientist Promega Corporation verview Brief overview of basic RNA/DNA chemistry. verview of total and poly(a+) RNA isolation. Discuss
DNA and RNA quantification: fast and simple with PicoGreen dsdna and RiboGreen RNA quantification reagents Fluorescence intensity on Infinite F2 and Infinite M2 Introduction DNA quantification Detection
1 DIAGNOSTICS MOLECULAR BIOLOGY MLSC 4050 Professor Labiner Module 1 Lecture 4: Nucleic Acid Extraction Nucleic Acid Extraction: Objectives 2 Students will be able to: 1. List the types of specimens that
Cat. # 9766 For Research Use TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Storage and shipping... 3 IV. Preparation
User Manual SYBR Advantage qpcr Premix User Manual United States/Canada 800.66.566 Asia Pacific +.650.99.7300 Europe +33.(0).3904.6880 Japan +8.(0)77.543.66 Clontech Laboratories, Inc. A Takara Bio Company
Cat. # RR091A or Research Use SYBR Premix DimerEraser (Perfect Real Time) Product Manual Table of Contents I. Description... 3 II. Principle... 3 III. Components... 4 IV. Storage... 5 V. eatures... 5 VI.
Table of Contents I. Description... 2 II. Kit Components... 2 III. Reagents not supplied in the kit... 3 IV. Equipment required... 3 V. Storage... 3 VI. References... 3 VII. Principle... 4 VIII. Features...
The Isolation and Sequence Analysis of the Cryptochrome (Cry1) Gene From a Petunia hybrida Plant By Jenalyn Quevedo Biology 115L June 6, 2005 1 Abstract The blue-light photoreceptor cryptochrome (cry1)
QuickExtract FFPE RNA Extraction Kit Cat. Nos. QFR82805 and QFR82050 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). www.epicentre.com
Applied Biosystems Real-Time PCR Rapid Assay Development Guidelines Description This tutorial will discuss recommended guidelines for designing and running real-time PCR quantification and SNP Genotyping
3.1.4 DNA Microarray Technology Scientists have discovered that one of the differences between healthy and cancer is which genes are turned on in each. Scientists can compare the gene expression patterns
STUDY OF VNTR HUMAN POLYMORPHISMS BY PCR Ref. PCR1 1. OBJECTIVE OF THE EXPERIMENT The objective of this experiment is to introduce students to the principles and practice of Polymerase Chain Reaction (PCR)
Quantitative analysis of PCR fragments with the Agilent 2100 Bioanalyzer Application Note Odilo Mueller Abstract This application note describes how the Agilent Technologies 2100 bioanalyzer can be used
For Research Use Premix Ex Taq (Probe qpcr) Product Manual Table of Contents I. Description... 3 II. Principle... 4 III. Components... 5 IV. Materials Required but not Provided... 5 V. Storage... 5 VI.