NIA Mouse cDNA Project FAQ

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• How can I obtain NIA Mouse cDNA Clone?
• Why I was unable to obtain NIA Mouse cDNA Clone from ATCC even though it has ATCC number?
• Where can I find NIA Mouse cDNA Library Vector Information?
• What is the NIA Mouse cDNA clone cloning direction?
• How can I view NIA Mouse cDNA Clone Set(s) verification results?
• How can I download Gene lists of NIA 15k and NIA 7.4k?
• How often Gene ID files will be updated?
• How can I obtain Gene Ontology Information of NIA Clone Sets?
• Where can I download published Microarray data?
• How can I find NIA EST Clones which are representing my gene(s) of interest?
• How can I find NIA Clones which were picked by Mammalian Gene Collection as potential full length Open Reading Frame (ORF) clone?
• How can I obtain NIA Clone Set(s)? How much does each one cost?
• How can I view Alpha Clusters information published in Development Paper?
• I am interested in some clones from NIA 15k and NIA 7.4k Mouse cDNA Clone Sets. How can I obtain individual clones of NIA 15k and NIA 7.4k Clone Sets?
• We have noticed some of the genes are human,rat,arabidopsis etc. What is the relationship of the mouse clone to human,rat or arabidopsis gene?. What similarity is there in sequence, gene function or other?. What is the process of assigning the gene id's to the sequences?.
• Is there any way to search the list of genes for a given gene product, for example "connexin43"?
• We are using NIA 15k Clone Set in our lab. I am looking for "H3027H10" clone sequence. When I look at your web site i found three different sequences (i.e H3027H10-3, H3027H10-5, G0110A02-3). Why is this?. If i click on one these seqnames i get a list of search sequence,masked sequence and trimmed sequence. What do these different sequences mean?. Which sequence belongs to H3027H10 clone?
• How to search the database with general keywords (e.g. apoptosis) to obtain an appropriate list of genes?
• I didn't find my question in the FAQ page. What should i do?
• We would like to check PCR product sizes as part of our Quality Control. Where can i find insert sizes of the NIA Mouse Clone Set Clones?
• How are the NIA Mouse cDNA Clones numbered?
• We are interested in obtaining NIA Mouse cDNA Clone Sets. Could you tell me from which lab you receommend I try to get it (for a reasonable fee)? Also could you tell me whether there is a list of papers that have been published using this clone set?
• I wish to use NIA Mouse 15 k microarray clones but I have one doubt: (1) Are these clones spotted on to a chip so that I can directly use them for probing with my RNA samples? If yes where can I get the micro array chip?.
• Can you please tell me what primers were used to obtain the 3' and 5' sequences. For example, was M13 Forward used to obtain 5' sequences for both sets, etc. Also, were the sequencing templates plasmid DNA or PCR products?

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1. How can I obtain NIA Mouse cDNA Clone?

NIA Clones are available from American Type Culture Collection (ATCC) (URL: http://www.atcc.org/SearchCatalogs/MolecularClones.cfm). You need an ATCC number to order a clone from ATCC. Please use the following Clone Search page http://lgsun.grc.nia.nih.gov/cDNA/Obtain-NIA-cDNA-Clone.html to find out ATCC number. If you click on the ATCC number link it will take you to ATCC clone ordering page.

2. Why I was unable to obtain NIA Mouse cDNA Clone from ATCC even though it has ATCC number?

ATCC might be in the process of making submitted clones available to researchers for ordering.

3. Where can I find Vector information of NIA cDNA Library?

You can find Vector information in the description field of NIA Clone Sequence GenBank Record.

4. What is the NIA Mouse cDNA clone cloning direction?

All NIA mouse cDNAs are cloned unidirectionally into SalI/NotI site in either pSPORT1 or pCMVSPORT6 vector. The direction is SalI -> 5'-cDNAs-3' -> polyA -> NotI

5. How can I view NIA Clone Set(s) verification results?

You can view NIA Clone Set(s) verification results at the following web sites. NIA Mouse 15k Clone Set:  http://lgsun.grc.nia.nih.gov/cDNA/PublicSeqVerify.html NIA Mouse 7.4k Clone Set: http://lgsun.grc.nia.nih.gov/cDNA/7kverify.html

6. How can I download Gene lists of NIA 15k and NIA 7.4k?

NIA Clone Set(s) GeneID files are available at the following web sites. NIA Mouse 15k Clone Set: http://lgsun.grc.nia.nih.gov/download.html NIA Mouse 7.4k Clone Set: http://lgsun.grc.nia.nih.gov/downloadnew.html

7. How often Gene ID files will be updated?.

For each new NCBI Mouse UniGene build, NIA Clone Sets GeneID files will be automatically updated.

8. How can I obtain Gene Ontology Information of NIA Clone Sets?.

You can use NIA Clone Set Gene Ontology Term Finder tool to obtain Gene Ontology Information of NIA Clone Sets. NIA Clone Set Gene Ontology Term Finder Tool is available at: http://lgsun.grc.nia.nih.gov/cDNA/NIA-CloneSet-GoTermFinder.html You can search for specific Gene Ontology terms. If you want to download all the Gene Ontology terms please don't enter any text in the text boxes.

9. Where can I download published Microarray data?.

You can download Laboratory of Genetics,NIA published Microarray data at the following web site. http://lgsun.grc.nia.nih.gov/microarray/data.html

10. How can I find NIA EST Clones which are representing my gene(s) of interest?.

You can find NIA cDNA Clones which are representing your gene(s) of interest in the following different ways 1. Blast Search   URL: http://lgsun.grc.nia.nih.gov/cDNAblast/ If you have gene(s) accession number(s) please follow the following five steps. Step 1: Select blast program to use.            If you have protein sequence accession(s) please select "tblastn" as blast program.            If your have nucleotide sequence accession(s) please select "blastn" as blast program. Step 2: Please select database against which you want compare your sequence Step 3: Please change data in the selection box before "search" button to "Accession or GI" Step 4: Please enter your gene accession number(s) in the text box. If you many accession numbers in a file you can upload gene(s) accession number(s) file by clicking "Browse" button Step 5: You can click on the "Search" button to perform blast search Note: If you don't have accession number you can get accession number by entering your gene symbol on the following page. http://www.ncbi.nlm.nih.gov/LocusLink/. Please select organism as "Mouse" and enter your gene symbol in the "Query" text box. When you click on Locus ID link, which is representing your gene of interest, it will show you all the mRNA sequence(s) accession numbers please select one of them and enter on the blast page If you have gene(s) sequence in fasta format please follow the following four steps. Step 1: Select blast program to use.         If you have protein sequence(s) fasta information please select "tblastn" as blast program.         If your have nucleotide sequence(s) fasta information please select "blastn" as blast program. Step 2: Please select database against which you want compare your sequence Step 4: Please enter your gene(s) sequence information in the text box. If you many sequences you can upload your gene(s) sequence fasta file by clicking "Browse" button Step 5: You can click on the "Search" button to perform blast search 2. NIA Mouse cDNA Clone Availability Search Tool (http://lgsun.grc.nia.nih.gov/cDNA/Obtain-NIA-cDNA-Clone.html) You can search by NCBI Mouse UniGene ID You can get Mouse UniGene ID by entering your gene symbol on http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm page. You can search by Gene Symbol You can enter your gene symbol to search NIA Mouse cDNA Clone database. You can enter partial string of the gene symbol if you would like search for NIA Mouse cDNA Clone that are representing genes with gene symbol name starting with your input string.

11. How can I find NIA Clones which were picked by Mammalian Gene Collection (MGC http://mgc.nci.nih.gov) as potential full length Open Reading Frame (ORF) clone?.

Please click http://lgsun.grc.nia.nih.gov/cgi-bin/NIA-MGC-FullLength-Clones to view NIA Mouse cDNA contribution to MGC. This web page will be updated every day.

12. How can I obtain NIA Clone Set(s)?. How much does each one cost?

NIA Clone sets are available from designated centers in United States, Canada, Europe and Japan. Please contact designated center about clone set price. To get more information about clone sets please visit the following web sites. For NIA 15k Clone Set: http://lgsun.grc.nia.nih.gov/cDNA/15k.html For NIA 7.4k Clone Set: http://lgsun.grc.nia.nih.gov/cDNA/NIA_7_4k.html

13. How can I view Alpha Clusters information published in Development Paper?

Please visit the following web page to view Alpha Clusters information published in Development 127, 1737-1749 http://lgsun.grc.nia.nih.gov/Alpha/index.html

14. I am interested in some clones from NIA 15k and NIA 7.4k Mouse cDNA Clone Sets. How can I obtain individual clones of NIA 15k and NIA 7.4k Clone Sets?

Individual clones of NIA Mouse 15k and NIA Mouse 7.4k clone sets are available from ATCC (http://www.atcc.org). You can find ATCC number for your NIA 15k or NIA 7.4k clone of interest by using http://lgsun.grc.nia.nih.gov/cDNA/Obtain-NIA-cDNA-Clone.html web page.

15. We have noticed some of the genes are human,rat,arabidopsis etc. What is the relationship of the mouse clone to human,rat or arabidopsis gene?. What similarity is there in sequence, gene function or other?. What is the process of assigning the gene id's to the sequences?

We compared NIA Mouse cDNA Clone Set sequences against NCBI 'nt' and NCBI Mouse RefSeq sequences. We store top 20 hits for each sequence in our database. We developed internal clone annotation alogorithm which will pick the best hit for the sequence. It will eliminate Genomic and other hits. If there is no mouse best hit then it will choose other species best hit.

16. Is there any way to search the list of genes for a given gene product, for example "connexin43"?

You can search NIA Mouse cDNA database using your gene product name for ex: connexin43 on NIA Mouse cDNA Clone Availability web page. Please enter your gene product name in the text box and select "NCBI Mouse UniGene Gene Symbol". Current gene symbol in the Gene Symbol(s) column will be in red color.

17. We are using NIA 15k Clone Set in our lab. I am looking for "H3027H10" clone sequence. When I look at your web site i found three different sequences (i.e H3027H10-3, H3027H10-5, G0110A02-3). Why is this?. If i click on one these seqnames i get a list of search sequence,masked sequence and trimmed sequence. What do these different sequences mean?. Which sequence belongs to H3027H10 clone?

H3027H10 (daughter) clone was rearrayed from G0110A02 (parent) clone. We sequenced daughter clones of NIA 15k and NIA 7.4k in both directions (3' -> 5'(3prime) and 5' -> 3' (5prime)). We verified NIA 15k clone (H3027H10) identity by comparing H3027H10-3 (3 prime sequence of daughter) and G0110A02-3 (3 prime sequence of parent). When the clone is verified you can use either daughter clone sequence (H3027H10-3 or H3027H10-5) or parent clone sequence (G0110A02-3). H3027H10-3 means sequence obtained from sequencing read of H3027H10 clone from 3' -> 5' direction. H3027H10-5 means seqeunce obtained from sequencing read of H3027H10 clone from 5' -> 3' direction. We receive the following three different sequences from sequencing of a clone. Raw Sequence: This is raw sequences we receive from sequencer Trim Sequence: The bases for this sequence were called by PHRED with an average 97% accuracy using the same trace file as was used to call 'searchseq'. This data may be longer, but less accurate than the 'searchseq'. We attempt to trim the vector, but not the Not1 and PolyT from this sequence. Search Sequence: The trace file for this sequence was read by PHRED and the bases were called with an average 99% accuracy. We attempt to trim the vector, Not1 site and PolyT from this sequence. All data processing e.g. RepeatMasking is done using this sequence Masked Sequence: We used repeat masker to mask search sequence. This is final sequence after repeats were masked. We are using this sequence for blast searches.

18. How to search the database with general keywords (e.g. apoptosis) to obtain an appropriate list of genes?

Currently you can search NIA 15k and NIA 7.4k Mouse cDNA Clone Sets using general keywords. You can use NIA Clone Set Gene Ontology Term Finder tool to obtain Gene Lists which have your keyword as function name. NIA Clone Set Gene Ontology Term Finder Tool is available at: http://lgsun.grc.nia.nih.gov/cDNA/NIA-CloneSet-GoTermFinder.html You can search for specific Gene Ontology terms. If you want to download all the Gene Ontology terms please don't enter any text in the text boxes.

19. I didn't find my question in the FAQ page. What should I do?

If your question was not answered in the FAQ page please send an email to Webmaster with the details.

20. We would like to check PCR product sizes as part of our Quality Control. Where can I find insert sizes of the NIA Mouse Clone Set Clones?

We don't have PCR product size information for NIA Clone Sets. You can visit the following NIA Clone Set distribution centers web sites to view NIA Clone Set Clones PCR product sizes NIA Mouse 15k Clone Set http://www.rtc.riken.go.jp/DNA/HTML/mouse15k/15kpict.html http://www.geneservice.co.uk/products/cdna/datasheets/NIA/gelimages.jsp http://array.mc.vanderbilt.edu/cDNA/NIA_15K/images_nia15k.vmsr NIA Mouse 7.4k Clone Set http://www.rtc.riken.go.jp/DNA/HTML/mouse15k/7_4kpict.html http://array.mc.vanderbilt.edu/Pages/Images/NIA_7.4K/nia_7.4K_pcr_images.html

21. How are the NIA Mouse cDNA Clones numbered?

Clone Names: All NIA Mouse cDNA clones, including ones that had been done in our laboratory before moving to NIA, have been named in the following manner. As an example of NIA naming conventions, a clone might be named J0054A09. The first part of the name (one letter and four digits), in this example J0054 gives the serial number of the plate where the clone can be found. This final part of the name (one letter and two digits), in this example A09 gives the well position of the clone in the plate. (Row "A" Column "09" on the plate) Sequence Names: Sequences obtained from the 5.-end of cDNA clone (e.g., H3078A10) are named with .-5. (e.g., H3078A10-5), whereas the sequences obtained from the 3.-end of cDNA clone are named with .-3. (e.g., H3078A10-3). When these two sequences were overlapped and assembled by Phred/Phrap, the assembled sequences are named with .-0. (e.g., H3078A10-0).

22. We are interested in obtaining NIA Mouse cDNA Clone Sets. Could you tell me from which lab you receommend I try to get it (for a reasonable fee)? Also could you tell me whether there is a list of papers that have been published using this clone set?

NIA Mouse clone sets (NIA 15k & NIA 7.4k) have been made freely available to the academic and other non-profit organizations. However, since our laboratory does not have a capacity to send otu the clone sets to individual laboratories, we have made an arrangement with laboratories/centers (distribution centers) who kindly agree to redistribute the cDNA Clone Sets. Although we have shipped the clone sets to the distribution centers for free of charge, the centers will be allowed to charge nominal fees in order to recover costs of materials, shipping and handling. Neither our laboratory nor NIH will not receive any monetary or material returns from the distribution centers. The centers do not have an obligation to redistribute the clone set. The details about this arrangement and the contact information of the distibution centers is available at the following web sites. NIA Mouse 15k Clone Set: http://lgsun.grc.nia.nih.gov/cDNA/15k.html NIA Mouse 7.4k Clone Set: http://lgsun.grc.nia.nih.gov/cDNA/NIA_7_4k.html Publications/Presentations of research data using NIA Mouse 15k Clone Set EDA targets revealed by skin gene expression profiles of wild-type, Tabby and Tabby EDA-A1 transgenic mice Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray Microarray analysis of somitogenesis reveals novel targets of different WNT signaling pathways in the somitic mesoderm Plac8 and Plac9, novel placental-enriched genes identified through microarray analysis Microarray and suppression subtractive hybridization analyses of gene expression in pheochromocytoma cells reveal pleiotropic effects of pituitary adenylate cyclase-activating polypeptide on cell proliferation, survival, and adhesion Identification of a novel set of genes regulated by a unique liver X receptor-alpha -mediated transcription mechanism Distinct gene expression profiles characterize the histopathological stages of disease in Helicobacter-induced mucosa-associated lymphoid tissue lymphoma Analysis of the PC12 cell transcriptome after differentiation with pituitary adenylate cyclase-activating polypeptide (PACAP) Identification, molecular characterization, and tissue expression of OVCOV1 Global analysis of stress-regulated mRNA turnover by using cDNA arrays AF5, a CNS cell line immortalized with an N-terminal fragment of SV40 large T: Growth, differentiation, genetic stability and gene expression Coupling of GnRH concentration and the GnRH receptor-activated gene program Inference of Genetic Network Using the Expression Profile Time Course Data of Mouse P19 Cells Expression profiling analyses of gonadotropin responses and tumor development in the absence of inhibins Gonadotropin-releasing hormone receptor-coupled gene network organization Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells Identification of a Novel Set of Genes Regulated by a Unique LXR-Mediated Transcription Mechanism DNA Microarray Analysis of Region-Specific Gene Expression in Mouse Epididymis Microarray analysis of Sparc null mouse lenses reveals a role for haemoglobin in lens biology? Development of a highly specialized cDNA array for the study and diagnosis of epithelial ovarian cancer Retinoid X Receptor {alpha} Regulates Glutathione Homeostasis and Xenobiotic Detoxification Processes in Mouse Liver.  Developmentally regulated expression of the regulator of G-protein signaling gene 2 (Rgs2) in the embryonic mouse pituitary  Gene expression profiling of cathepsin D, metallothioneins-1 and -2, osteopontin, and tenascin-C in a mouse spinal cord injury model by cDNA microarray analysis  Uterine extracellular matrix components are altered during defective decidualization in interleukin-11 receptor alpha deficient mice  Vitamin D treatment of senescence accelerated mice (SAM-P/6) induces several regulators of stromal cell plasticity  Microarray gene expression analysis of the Fob3b obesity QTL identifies positional candidate gene Sqle and perturbed cholesterol and glycolysis pathways  Gene expression changes during cataract progression in Sparc null mice: differential regulation of mouse globins in the lens.  Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Developmentally regulated expression of the regulator of G-protein signaling gene 2 (Rgs2) in the embryonic mouse pituitary A time course microarray study of gene expression in the mouse lacrimal gland after acute corneal trauma. Global analysis of the expression patterns of transcriptional regulatory factors in formation of embryoid bodies using sensitive oligonucleotide microarray systems. Lsh, an epigenetic guardian of repetitive elements. Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing. Genetic aspects of melatonin biology. A global view of gene expression in the preimplantation mouse embryo: morula versus blastocyst. Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease. Genetic regulation of endotoxin-induced airway disease.

23. I wish to use NIA Mouse 15 k microarray clones but I have one doubt: (1) Are these clones spotted on to a chip so that I can directly use them for probing with my RNA samples? If yes where can I get the micro array chip?.

We do not provide microarrays. You will probably have two options. If you know any microarray facility nearby or at your institute, please check whether they have the NIA mouse cDNA clone set (Tanaka TS, et al., Proc. Natl. Acad. Sci. USA, 97: 9127-9132. [PMID: 10922068];38. Kargul GJ, et al., Nature Genetics, 28:17-18. [PMID: 11326268]; VanBuren V, et al. Genome Res 12: 1999-2003. [PMID: 12466305]) and have prepared the arrays. The clone sets have been made available to the research community. Please check the following web sites for the details. NIA Mouse 15k Clone Set NIA Mouse 7.4k Clone Set We have worked with Agilent to develop 60-mer oligonucleotide microarrays on glass-slide (Carter M. et al., Genome Res.13: 1011-21. [PMID: 12727912]), which is commercially available from Agilent. This array, called Mouse Development Arrays, roughly corresponds to the NIA mouse 15K and 7.4K cDNA clone sets. For more information please visit Mouse Development Oligo Array web page.

24. Can you please tell me what primers were used to obtain the 3' and 5' sequences. For example, was M13 Forward used to obtain 5' sequences for both sets, etc. Also, were the sequencing templates plasmid DNA or PCR products?>

We used the following primers to obtain 3' and 5' sequences. 3' Sequence: -21M13Forward (3') 5' TGTAAAACGACGGCCAGT 3' 5' Sequence: M13 Reverse (5') 5' CAGGAAACAGCTATGACC 3' Sequencing templates were plasmid DNA.