M

Mad cow disease, 157 Master mixes, 91 Mathematical model for DD, see DD, mathematical model for coverage mDAZL RNA binding protein, 300, 302,311 Melancholic depression, 279-280 Microarray and DD combined, see Vertical arrays Microarrays, 4 problems, 4 publication numbers, 25 mob-5, see IL-24 Modified single-strand conformational polymorphism (mSSCP), 163-165 mRNA binding, see RNA binding proteins mRNA polymorphism detection, 279-285 mRNA purification, see RNA isolation, mRNA polyA+ RNA...

Cc Cc

(Note G T substitutions in the 3rd base from 3'end) (Note G T substitutions in the 3rd base from 3'end) (Note G T substitutions in the 3rd base from 3'end) Two bases covering the restriction site (with mismatch, suitable for Rsal and HaeIII cleaved DNA) Extended T-primers (TE-primers) 5'-cgc agt cgg tac (t)i3 AG AA AT AC And so on, all extension variants except those with T in the distal position. 25. 10X polynucleotide kinase buffer (500 mM Tris-HCL, pH 7.6, 100 mM MgCl2, 10 mM spermidine, 10...

Rosana Risques Gaelle Rondeau Martin Judex Michael McClelland and John Welsh

Vertical arrays are microarrays that have complex mixtures of nucleic acids as array elements, and that are hybridized with single sequence probes. Like dot blots, many different experiments can be spotted on a single vertical array, allowing single genes to be compared across many conditions. Vertical arrays have two additional advantages over dot blots. First, they are printed on glass slides, allowing the use of low-volume, high-concentration hybridization reactions. Second, they can be made...

Reamplification of cDNA Fragments and Northern Blotting

Extraction of cDNA Fragments From the Dry Sequencing Gel Select positive bands based on difference between the control and test samples (see Note 4). Align the film with the dried gel on the three needle holes. Place small clips along the edge of the film and gel to keep the film in place. Use a 23-gage needle to puncture through both the film and the gel at four corners of the positive bands. After the bands are excised from the gel, cut the gel fragments into smaller pieces before they...

Differentially Expressed Genes Associated With Hepatitis B Virus HBx and MHBs Protein Function in Hepatocellular

HBx and MHBs' products from hepatitis B virus-DNA (HBV-DNA), which become transcriptional transactivators of cellular and viral genes, are known to play causative roles in the development of hepatocellular carcinoma (HCC). However, the biomolecular mechanism(s) for their roles in hepatocarcinogenesis in vivo remain poorly understood. To identify authentic cellular genes involved in HBx and MHBsl-transactivated carcinogenesis, we used mRNA differential display polymerase chain reaction (DD-PCR)....

Reamplification Using Extended Primers

The next steps describe the reamplification of cDNAs that have been recovered from the mSSCP gel. This involves reamplification for five cycles in the presence of a modified version of the random 10mer and (dT)12 primers used in the original display reaction. Primers have been modified to include EcoRI restriction sites to facilitate cloning of the amplified fragment. 1. Spin the precipitated cDNA recovered from the mSSCP gel (Subheading 3.5.2.) at 10,000g for 25 min at 4 C. 2. Wash the pellet...

Notes

To avoid detecting differences that were irrelevant to p53 induction, two sets of cells, one washed with media containing tetracycline and the other without tetra-cycline were prepared. After incubating the cells with fresh media either with (no induction) or without (p53 induction) tetracycline, RNA samples were extracted at 4 and 8 h thereafter (21). 2. To avoid pipetting errors as much as possible, core FDD mixes containing everything except, cDNA and arbitrary primers were made, before...

References

Lin, Y., Ma, W., and Benchimol, S. (2000) Pidd, a new death-domain-containing protein, is induced by p53 and promotes apoptosis. Nature Gen. 26, 124-127. 2. Oda, E., Ohki, R., Murasawa, H., et al. (2000) Noxa, a BH3-only member of the Bcl-2 familiy and candidate mediator of p53-induced apoptosis. Science 288, 1053-1058. 3. Oda, E., Arakawa, H., Tanaka, T., et al. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 102, 849-862....

Info

Because the resulting cDNAs are fluorescently labeled, the use of a fluorescent imager scanner is required for this technology. Here the FMBIO laser imager series (MiraiBio, Alameda, CA) is recommended for digital acquisition of the cDNA profiles. Although this is the recommended imager, other fluorescent scanners, such as the Typhoon (Amersham Biosciences, Piscataway, NJ) and FLA-5000 (FUJIFILM Medical Systems, Stamford, CT) can also be used for FDD with similar sensitivity. Another option for...

A B C D E F G H

Arrangement of samples in a 96-well plate for loading on a denaturing gel with an 8-channel syringe. Each set of six PCR reactions (samples 1-3 represent RNA no. 1, samples 4-6, RNA no. 2) is loaded as indicated. For simplicity, only first two columns are shown. 5. For PCR, use the following cycling program initial incubation at 94 C for 3 min, then 94 C for 30 s, 40 C for 2 min, and 72 C for 30 s (40 cycles). Extend the last cycle by a 5-min incubation at 72 C. Store the plate at -20...

Double Stranded cDNA Synthesis

This protocol describes a standard procedure of double-stranded cDNA synthesis that uses RNAseH DNA polymerase I DNA ligase cocktail for synthesis of the second cDNA strand. Any commercially available kit that utilizes the same principle (such as Marathon kit by BD Biosciences Clontech) can be used at this step, except that the primer for first-strand synthesis provided in the kit should be substituted for TRsa. 3.2.1. First-Strand cDNA Synthesis 1. To 5 L RNA solution in water (0.03-3 g of...

Preface

Since the first edition of this book dedicated to differential display (DD) technology was published in 1997, we have witnessed an explosive interest in studying differential gene expression. The gene-hunting euphoria was initially powered by the invention of DD, which was gradually overtaken by DNA microarray technology in recent years. Then why is there still the need for second edition of this DD book First of all, DD still enjoys a substantial lead over DNA microarrays in the ISI citation...

Confirmation of Differential Gene Expression by Northern Blot

The confirmation of the differential gene expression found by the FDD procedure by Northern blotting is very important because the method is extremely sensitive and details can be obtained about the exactly mRNA size of the screened cDNA fragments. The Northern blot can be performed by using the HotPrime DNA labeling kit, following the standard procedure (7). The HotPrime DNA labeling Kit is up to 10 times better in labeling efficiency than traditional Random-Prime kit. This high labeling...

RT Reaction and FDDPCR

5X Reverse Transcription (RT) buffer 125 mM Tris-HCl, pH 8.3, 188 mM KCl, 7.5 mM MgCl2, 25 mM DTT (GenHunter) (see Note 1). 2. MMLV Reverse Transcriptase (100 U (L) (GenHunter) (see Note 1). 3. dNTP Mix (2.5 mM) (GenHunter) (see Note 1). 4. H-TnV anchor primer (V A, C, G) (2 (M) (GenHunter) (see Note 1). 5. R-H-TnV anchor primer (V A, C, G) (2 (M) (GenHunter) (see Note 1) (Rhodamine-labled primers are light sensitive). 6. H-AP 13mer primers (1-160) with 50-70 GC content (2 (M) (GenHunter) (see...

Electrophoretic Separation

Fluorescent one-color sequencing apparatus (ALFexpressll, Amersham Biosciences, London). 2. 0.5 mm thick denaturing 4 T 3 C polyacrylamide gel (separation distance 20 cm). 3. Standardized electrophoretic conditions (55 C, 1500 V, 60 mA, 50 W) and sampling interval of 2 s. 4. An internal Cy5-labeled size standard series (633 bp, 647 bp, 680 bp). 5. An external molecular weight ladder (ALF Sizer 50-500 bp, Amersham Biosciences).

Results

Identification of IL-24 as an Immediate Target Gene of Oncogenic h-ras Using DD screening with rational primer designs (3), two paradigms, one with Rat-1 iRas cells containing an IPTG inducible oncogenic h-ras, the other with oncogenic h-ras transformed Rat-1 cells before and after treatment of a MAP kinase kinase inhibitor PD98059, were set up for the systematic screening of oncogenic ras target genes. After screening through 90 combinations of primers, a total of 14 ras inducible genes...

Arthur B Pardee

Dana Farber Cancer Institute, Boston MA 2006 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or...

B

Distance calculation Trace comparison Fig. 1. Schematic representation of the phases included in the current interactive DD data analysis protocol. (A) Peak assessment phase detects and quantifies valid peaks in the given electrophoretic trace data and exports size-ordered peak lists for subsequent computational analysis (see Note 4). (B) In the trace comparison phase, peak complexes are modeled and aligned, and score-ranked comparison results are created for confirmation and further analysis...

G G A C A G G A C A

Site selection phylogenetic footprinting Fig. 3. Conservation of Xbpl binding sites in the promoters of genes identified by DD. Below are alignments of the CLN3, CLN1, and CLB2 promoters from yeast species highly related to Saccharomyces cerevisiae Saccharomyces bayanus, Saccharo-myces castelli, Saccharomyces kudriavzevii and+ Saccharomyces mikatae. Conserved positions of the Xbpl binding site consensus are depicted in black boxes positions conserved among more than 50 of the listed binding...

Sequence Analysis of the Differentially Amplified Bands

The sequences from all of the clones (trimmed of vector and primer sequences) are compared and assembled into contiguous sequences (contigs) using sequence assembly software like Sequencher (Genecodes, Ann Arbor, MI). Each contig is assembled from the overlapping sequences of the multiple clones of the same differentially amplified band. Some contigs are also assembled from the sequences of different bands generated in distinct RT-PCR reactions with different arbitrary primers, indicating that...

S Hi

Problem for two-base anchored primers ending with the 3' T, and reduces the number of reverse transcription reactions from 12 to 3 per RNA sample. 2. It has been observed that the 35S-labeled nucleotide originally used for DD would leak through PCR reaction tubes (especially when thin-walled tubes are used) and 33P labeled nucleotide was recommended as the best alternative (9). 33P is not only safer to use, but also gives better sensitivity as compared to 35S. 3. For the RT reaction, the...

Lcr I

Vertical array detection of differential regulation in response to the addition of serum to serum-starved fibroblasts. The top row shows an LCR in which a sequence homologous to AA42873 is absent the (red) fluorescent signal is from the positive hybridization control. The bottom row shows hybridization at t 0 and t 20 min (violet), but not at 4 h using an LCR in which the transcript is sampled. Fig. 1. Vertical array detection of differential regulation in response to the addition of...

Isolation of Total Cellular RNA

Prokaryotic mRNAs are short-lived, with a half-life on the order of minutes in fast-growing species. The procedures for RNA extraction must, thus, be very rapid. Techniques of cell disruption based on a lysozyme treatment to digest bacterial cell walls are too slow (tens of minutes to hours) and lead to highly degraded mRNA. The protocol for the isolation of total cellular RNA uses a physical disruption of bacterial cells with zirconia beads in a bead beater in the presence of the denaturing...

Ccatgg Tcga 22 Tcga Ccatgg

GenEST uses the begin tag to search for the tag sequence in the cDNA data, which are contained in the input files in FASTA format. If such a tag is found, it will continue its search for a matching end tag. This search action is executed in both directions for all begin end tag combinations. 9. To execute the search, type in a DOS window C genest gt ruby src genest -c NTtag.txt -o TDFout.txt EST.txt -o TDFout.txt - indicates the output file, which will contain a list of the found virtual...