title: TAGC_H.Sapiens_9K_20041223
status: Public on Mar 01 2007
contact.city: angers
contact.first: Jean-Fred
contact.last: Fontaine
contact.email: jean-fred.fontaine@univ-angers.fr
contact.address: 4 rue Larrey
contact.country: France
contact.institute: INSERM
contact.phone: +33 (0)2 41 35 33 14
organism: Homo sapiens
manufacture_protocol: Introduction:
  Nylon microarrays are a thumbnail version (of glass-slide format) of the classical macro-membranes (1-3), allowing a larger probe density compatible with pan-genomics approaches. These microarrays have been developped by Konan Peck (4). They have the advantage of an easy setup in an academic lab at very low cost, to use classical protocols of Molecular Biology, to be reusable, and to use an universal reference. Moreover, they use smaller amounts of samples without any amplification step (5): typically microarrays can be hybridized with 1 to 5µg of total RNA. Major drawbacks of this technique are a lower density of spots than other microarrays (a maximum of 15,000 spots on a glass-slide) due to indirect scanning of radioactivity, strong spots overshining, and an independent hybridization of the sample and the reference. Typically, a Nylon microarray measurement consist in one hybridization to measure the spotted amount of probes (hybridization using an oligonucleotide complementary of a vector sequence present in all PCR products), and one hybridization with the sample. Final measurement is the ratio sample/spotted amount. This measurement is necessary due to the dependency of the signal to the spotted amount of probe (5-6). One key point of this technique is to spot a large amount of probe to have a good detection (5). Nylon microarrays can also be used with other types of probes such as short or long oligonucleotides (7).
  PCR:
  Oligonucleotides allowing PCR amplification of clones from various IMAGE (8-9) cDNA libraries have been designed. They allow embedded PCR (LBP1A/AS and LBP2A/AS) and the measurement of spotted amounts using an internal primer (LBP3 or LBP9). It is essential that the oligonucleotide used for spotted amount measurement is not one used for PCR amplification, otherwise primer dimers occurring during PCR amplification should be measured at the same time than specific probes: measurement would be wrong.
  Ten microliters of a bacterial suspension in water are added to 90µl of a PCR mix. The amplification is done by:
      * 94°C 6'
      * 40 cycles (94°C 30'', 55°C 40'', 72°C 1')
      * 72°C 10'
  One microliter of PCR is deposited on 1% agarose gel. Two hundreds microliters of PCR products are evaporated at 94° in the PCR machine and resuspended in 60µl of water.
  PCR product concentration should be between 150 to 300 ng/µl. Precise quantification of PCR products (if necessary) can be done directly from the gel. Methods using double-stranded DNA intercalating agents should be avoided as PCR generates double-stranded dimer of primers that are quantified at the same time. It is not necessary to purify the PCR products. PCR products showing wrong size, 2 bands or week should be discarded.
  Spotting :
  Precut Nylon membranes (Pall) are fixed on glass-slides in the robot using spray glue (3M). Spotting of PCR products in 384-well plates, is done using GMS417 (Affymetrix) ou BioGrid II (Matrix) robot. Spot to spot distance is 375µm, and each PCR product is spotted 4 times for greater amount spotted. Temperature is maintained at 18°C and humidity at 70% during the spotting process.
  After spotting, filters are deposited (spotted side up) onto a blotting paper saturated with denaturation buffer for 2 times 10', and on a blotting paper saturated with neutralization buffer for 2 times 10'. Filters are rapidly rinsed with  2xSSC, dried in an owen at 80°C for 2H, and irradiated with short UV during 1'30''. They can be stored during years.
  Oligonucleotide labelling & hybridization:
   One microliter of oligonucleotide (LBP3 or LBP9) at 1µg/µl is added to 24µl of the  oligonucleotide kinase mix. Incubate 10' at 37°C, 10' at 65°C in a waterbath, and purify on a G25 Sephadex column (Centrifuge the column for 2' at 2,500 rpm, load 100µl  of the kination reaction on top the column, centrifuge 4' at 1,000 rpm, keep the eluate), and count radioactivity.
  Prehybridize membranes in the hybridization solution containing 100µg/ml heering sperm DNA denaturated (by heating 10' at 100°C and quickly cooled on ice) during  4H (10H maximum) at 42°C. Add 50.000 cpm of the labelled oligonucleotide and hybridize during 10H at 42°C. Wash with the washing solution during 10' at room temperature, and for 5' at 42°C with preheated solution. Quickly rinse filters with 2xSSC, and dry them on blotting paper. Expose dry filters onto a screen in a cassette (Raytest/Fuji) for about  8H. Scan with radioimager (Fuji Bas5000) at 50µm resolution.
  Strip membranes in the heated washing solution  at 68°C in a waterbath during 3H, change solution each hour. Check for stripping by an exposure of 8H on an exposure screen. If some signal remains, try to strip the filters a second time.
  Many filters can be hybridized and washed together. In this case, use larger volumes.
  RNA labelling & hybridization :
    1 to 5 µg of total RNA are added to 8 µg dT25 (to saturate polyA tails), 0,3 ng spike RNA, and 13 µl of water. Incubate for 8’ at 70°C in a waterbath. Put in a dry block heater containing water at 70°C, and put the block in an owen at 42°C for 30’. Add 17 µl of labelling mix. Incubate at 42°C for 1H. Add 1µl of reverse transcriptase. Incubate one more hour at 42°C. Successively add 1µl SDS 10%, 1µL EDTA 0.5M, and 3µl NaOH 3M. Incubate 30’ at 68°C, to degrade mRNA. Incubate 15’ at room temperature. Add 10µl of Tris 1M, and 3µl of HCl 2M for neutralization. Add 2µG of dA80 and 2µg Cot1 DNA. Heat 5’ at 100°C, add 300µl of h of hybridisation buffer preheated at 65°C, and incubate for 2H30 at 65°C to saturate polyT tracks and repeats. It is not necessary to purify.
  Rinse spotted membranes with 2xSSC. Pre-hybridize with 2ml of hybridisation buffer containing 100µg/ml heering sperm DNA denaturated (by heating 10' at 100°C and quickly cooled on ice) for 6H at 68°C. Remove buffer and replace by the labelled cDNA. Hybridize at 68°C for 24H to 48H in a rotating owen.
  Quickly rinse membranes with hybridisation solution pre-heated at 68°C. Wash the membranes with a large volume of washing solution for 3H at 68° (Change solution each hour). Rinse with 2xSSC, and dry on blotting paper. Expose dry filters onto a screen in a cassette (Raytest/Fuji) for about 24H. Scan with radioimager (Fuji Bas5000) at 50µm resolution.
  Strip membranes in the heated dehybridation solution  at 85°C in a waterbath during 5H, and quickly rinse with 2xSSC. Control stripping by an exposure of 8H on an exposure screen. If some signal remains, try to strip the filters a second time. If this protocol does not works well, repeat this after a one month delay.
  References :
     1. Nguyen C, Rocha D, Granjeaud S, Baldit M, Bernard K, Naquet P, Jordan BR.. Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones. 1995 Genomics 29: 207–216.
     2. Zhao, N., Hashida, H., Takahashi, N., Misumi, Y. & Sakaki, Y. High-density cDNA filter analysis: a novel approach for large-scale, quantitative analysis of gene expression. 1995 Gene 156: 207–213.
     3. Piétu G, Alibert O, Guichard V, Lamy B, Bois F, Leroy E, Mariage-Samson R, Houlgatte R, Soularue P, Auffray C. Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array. 1996 Genome Research  6: 492-503.
  4. Chen JJ, Wu R, Yang PC, Huang JY, Sher YP, Han MH, Kao WC, Lee PJ, Chiu TF, Chang F, Chu YW, Wu CW, Peck K. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. 1998 Genomics. 51: 313-324.
     5. Bertucci F, Bernard K, Loriod B, Chang YC, Granjeaud S, Birnbaum D, Nguyen C, Peck K, Jordan BR. Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples. 1999 Human Molecular Genetics 8: 1715 –1722.
     6. Stillman BA, Tonkinson JL. Expression microarray hybridiza