Methods and reagents: Expression profiling using messenger RNA assays Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses the use of hybridization techniques for the quantitation of gene expression and a contaminant found when isolating His-tagged proteins. For details on how to partake in the newsgroup, see the accompanying box. Determining differences in gene expression between cell lines of developmentally related or diseased tissues, or between cells responding to various outside stimuli, has gained attention recently in the methods group. Apart from the generally accepted methods of differential cDNA library screening or differential display [1], netters have discovered some new tools based on the polymerase chain reaction (PCR). Expression profiling ******************** A quantitative method to measure gene activity, called multiplex messenger assay (MMA), can be performed by immobilizing a library of cloned cDNA onto filter membranes. Liquid bacterial cultures containing different cDNAs can be spotted onto filters in the form of an array, or can be transferred as bacterial colonies directly from agar plates onto the membrane. A complex mixture of labeled total RNA isolated from the cell line to be analysed is then used as probe against the immobilized cDNA targets. The relative amounts of mRNA hybridizing to each known target cDNA can then be determined by quantitating the signal observed within each spot on the X-ray film [2]. Reverse northern blots. Some netters familiar with this method were initially confused because someone referred to the technique as a `reverse northern' blot. It is considered to be reverse because target DNA is being probed with RNA, which is the reverse of what is normally done with a northern blot - RNA probed with DNA. While some think it is okay to keep the name of the technique within the slang terms used in molecular biology, others thought that the term is not at all intuitive, and that it is too archaic to be understood clearly. When people refer to northerns, it is also assumed that there would be transfer of RNA or some other material from a gel onto a membrane. In addition, the term `reverse northern' has been applied to other types of hybridization reactions and therefore has not been properly defined in the literature. Others agreed that a more original term should be selected and that perhaps expression profiling or multiplex messenger assay (MMA) would be better, although it is still not obvious from those names that a hybridization reaction is being performed. If done carefully, MMA can show the varying levels of transcription from different genes in a single experiment, and as well as showing the activity of known genes, the method can also reveal information about various transcripts without prior knowledge of their function or the sequence of the gene encoding them. Further, quantitating mRNA with this technique is possible without ever having cloned the cDNA into a vector. Reverse transcriptase PCR. In cases where messenger RNA is of low abundance, several variations of this theme have been described in which the probe is composed of a heterogeneous population of mRNA amplified using reverse transcriptase PCR (RT-PCR) in combination with in vitro transcription. For example, use of an oligonucleotide primer containing a region of polythymidine and the promoter sequence recognized by T7 RNA polymerase (RNAP) on the 5'-end allows the production of mRNA directly from amplified DNA, rather than from either cloned or genomic DNA. Antisense RNA. Instead of using the mRNA that was isolated directly from the cell line as a probe, the RNA probe is composed of radiolabeled antisense RNA (aRNA) that was generated by a reverse-transcription reaction on the total RNA into cDNA, followed by an amplification of the cDNA by the PCR, and then an in vitro transcription reaction by T7 RNAP, converting that cDNA into aRNA. One study claims that an 80-fold molar amplification of probe RNA can be achieved from nanograms of cDNA in this way [3]. Netters were quick to point out that the multiple enzymatic steps involved can possibly lead to problems if any of the reactions are less efficient for one type of RNA. This could affect the interpretation of the hybridization results. Care should also be taken when designing primers for such an experiment, as it was recently shown that T7 promoter primers of varying length are not equally efficient in transcribing aRNA from PCR-generated templates [4]. `Micro-reverse northerns'. A method similar to MMA was also recently published in which short fragments of the target DNA are tethered to a glass slide in microarrays and the hybridization reaction is performed with fluorescently labeled cDNA probes to obtain the expression profile [5]. One netter jokingly suggested that the term `micro-reverse northern' be adopted for this technique. Unfortunately, the high-end, sophisticated equipment needed to perform this type of experiment prohibits its use in most labs. If not already available, there could be a future market for prefabricated micro-zoo slides holding complete cDNA libraries for various animals, and perhaps for a cheap instrument to be used for the analysis of the fluorescent hybridization probes. More His-Tag problems ********************* During this past month, some netters were discussing more problems with co-purifying proteins when using immobilized metal-ion affinity chromatography (IMAC) for recovery of histidine-tagged fusion proteins. For a review on the use of His-Tags, see the Methods and reagents column in TiBS 20, 285-286 (1995). Malini R. Madiraju (malini@uthct.edu) recently complained about having a 25 kDa contaminant along with the expected 55 kDa fusion protein, and that repeated step gradients performed on the affinity column to separate the two proteins were unsuccessful. After much difficulty, this netter found that when the recombinant fusion protein is overexpressed in Escherichia coli BL21/pLysE or BL21/pLysS, the contaminant always co-purifies with the desired protein. Although one netter wrote that a protein obtained in a similar way was determined to be beta-galactosidase by searching a database with the sequence derived from a likewise contaminating protein, others felt that the 25 kDa contaminant is most likely chloramphenicol acetyltransferase (CAT). CAT is encoded by the chloramphenicol resistance gene of the pACYC184-derived plasmids pLysE and pLysS, commonly used to provide the antagonist to T7 polymerase for control of transcription in T7-based expression systems. Although it was not confirmed by Dr Madiraju that the culprit in her case was indeed the CAT enzyme, a recent study showed that the eluate from a nickel-NTA column was always contaminated with a protein of 25 kDa, no matter what concentration of imidizole was used for the elution. In addition, the contaminant persisted even when 1M NaCl and 0.5% Triton-X 100 were added to the equilibration and elution buffers. The 25 kDa protein was later confirmed to be CAT by N-terminal protein sequencing. That CAT is His-rich, having 21 His residues out of 219 amino acids, is not surprising [6]. Netters now suggest that chloramphenicol resistance should be avoided as the selection marker when synthesizing proteins to be purified by IMAC. One alternative is the more tightly regulated T7 polymerase expression system based on the bacteriophage lambda attenuated regulation at PL. Because the gene for T7 polymerase is maintained on a low copy number plasmid under strict regulatory control, and the selective marker for that plasmid is kanamycin resistance rather than chloramphenicol resistance, the induction of genes cloned downstream of the T7 promoter on any other compatible plasmid vector is achieved without the need for pLysE and pLysS [7]. References ********** [1] Liang, P. and Pardee, A. B. (1992) Science 257, 967-971 [2] Bernard, K. et al. (1996) Nucleic Acids Res. 24, 1435-1442 [3] Van Gelder, R. N. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1663-1667 [4] Baklanov, M. M., Golikova, L. N. and Malygin, E. G. (1996) Nucleic Acids Res. 24, 3659-3660 [5] Schena, M., Shalon, D., Davis, R. W. and Brown, P. O. (1995) Science 270, 467-470 [6] Oswald, T. and Rinas, U. (1996) Anal. Biochem. 236, 357-358 [7] Mertens, N., Remaut, E. and Fiers, W. (1995) Bio/Technology 13, 175-179 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/dec96.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1996. Methods and reagents: Expression profiling using messenger RNA assays. Trends in Biochemical Sciences 21(12):492-493. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************