Evaluation of Mouse Neural Stem Focused Array and Quality Control

To assess quality of the membranes one needs to evaluate the quality of the DNA, the accuracy of the sequence information, the quality of printing and the specificity of hybridization.

3.3.1. Quality Control of Printed Membranes

1. Observe the spots that have been stained by tracking day (0.001% bromophenol blue) by eye during printing (see Note 7).

2. Perform array hybridization and analysis by using a mixture of total RNA from mouse D3 ES cells and adult rat hippocampus that covers at least 90% of the genes in the chip (see Subheading 3.3.3.) and following procedures described under Subheading 3.2.

3. Perform data analysis (see Subheading 3.2.4.). Results are shown in Fig. 4. Gene expression scored as existing (positive) or absent (negative) shows a high repro-ducibility irrespective of the probe preparation technique used (Fig. 4C) even when different batches of RNA were used. All 10 blank spots were consistently negative, duplicates showed consistent levels of expression and virtually all positives remained positive.

3.3.2. Cross Hybridization Assessment

It is important to determine that arrayed genes can be detected in a specific fashion without cross hybridization.

1. Randomly select 17 genes to test the specificity of hybridization. For example, we used fgf4, Gcm2, Mtap1b, Mtab2, Tubb3, Prox1, NFL, NCAM2, Cst3, Ccng2, and CDKn1b, DNMT1, Cdh4, Itga6, Rpl13a, Actb, and GAPDH. In addition, spot some genes in duplicate or triplicate. The location of each gene in the array is bolded in Fig. 1.

2. Label probes by PCR in the presence of biotin-dUTP. Each reaction (10 ||L) contains 1 |L 10X PCR buffer, 150 |imol MgCl2, 0.8 |L buffer BN, 20 pmol primer, 1 |L 100X diluted specific gene inserted plasmid, 1 U RedTaq DNA polymerase, and 0.2 pmol biotin-dUTP.

3. Run PCR in 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final extension for 10 min at 72°C.

4. Take 3 | L of each labeled cDNA fragment to check probe quality by running it in 1.2% agarose gel (Fig. 5A).

5. Pool the labeled probe for each gene and perform an array (see Subheading 3.2.).

6. Perform data analysis (see Subheading 3.2.4.). As shown in Fig. 5B, the biotinlabeled probe of each gene identified only its corresponding arrayed gene fragment, suggesting limited or no cross-hybridization. This is clearly illustrated with FGF4, a member of the FGF family. A total of 22 members were spotted on the chip that share varying degrees of homology. The FGF4 probe gave a strong signal on the FGF4 spot and showed no cross-hybridization with any of other FGFs, suggesting a high specificity for this gene hybridization under our conditions. Moreover, the chip has a high quality in printing. All duplicates (FGF4 and NCAM2) and triplicates (Rpl13a, Actb, and GAPDH, underline in Fig. 5B) were evenly detected.

3.3.3. Assessments of Detection Sensitivity by Comparison of Labeling Protocols Between AmpoLabeling (LPR) Kit and Conventional MMLV RT Method

1. Choose a RNA source that expresses the majority of genes printed on the focused membranes. We used a mixture of RNA from mouse D3 ES cells and (0.6 |g) adult rat hippocampus (0.2 |g)/|L because PCR amplification could readily detect 234/260 genes in this pooled RNA.

2. Perform array analysis by using this pooled RNA to determine how many of the 260 arrayed genes are detectable using either standard MMLV RT or LPR protocol (see Subheading 3.2.).

3. Perform data analysis (see Subheading 3.2.4.). A representative imaging profile and results are shown in Fig. 4 (see Note 8).

Fig. 4. Quality control of the chips and comparison of results derived from moloney murine leukemia virus reverse transcriptase (MMLV RT) and linear polymerase reaction (LPR) or Ampolabeling methods. Total RNAs from mouse D3 ES cells and adult rat hippocampus were first isolated and then mixed together with a ratio of 3:1. This mixed RNA (0.8 ^g/membrane) was used to prepare probes using MMLV RT or LPR methods. Labeled probes were used to perform array hybridization. (A) Shows distinct

Fig. 4. Quality control of the chips and comparison of results derived from moloney murine leukemia virus reverse transcriptase (MMLV RT) and linear polymerase reaction (LPR) or Ampolabeling methods. Total RNAs from mouse D3 ES cells and adult rat hippocampus were first isolated and then mixed together with a ratio of 3:1. This mixed RNA (0.8 ^g/membrane) was used to prepare probes using MMLV RT or LPR methods. Labeled probes were used to perform array hybridization. (A) Shows distinct

3.3.4. Array Test for Specific Tissues

A real evaluation of quality of the array is to determine if the arrayed genes were selective and sufficient in number to differentiate between different cell populations.

1. Choose cell types. We examined embryonic stem cells (undifferentiated mouse D3 ES cells) that are developmentally closely related to neural cells and share many antigens (6,7), and fetal neural cells (rat E14.5 neural tubes) that consist of predominantly neural stem cells and neural progenitors (8-10). We included liver cells (derived from 6-mo-old male Sprague-Dawley rat) as an endodermal derivative that is distinct from the other two populations.

2. Isolate total RNA by using TRIzol.

3. Perform array analyses by a standard MMLV-RT labeling protocol (see Subheading 3.2.).

4. Perform data analysis (see Subheading 3.2.4.). As shown in Fig. 6A, the gene expression profile could be easily distinguished between these cell types. There were nine ES markers detected in mouse D3 ES cells that were absent or expressed at low/undetectable levels in neural cells or in liver (a list in Fig. 6C). Six of these ES markers were easily identified in the first row. Dnmt1 and Itga6 were located in rows 15 and 17, respectively, and appeared to be specific to ES cells though they were not initially included on the array as cell type specific markers. The relatively specific expression in D3 cells was confirmed by RT-PCR. As shown in Fig. 6B,C, Sox2 is present in D3 (ES cells) as well as E14.5 neural tubes (neural stem and progenitor cells) consistent with published data of Sox-2 expression in ES and neural stem cell populations (4,11). In addition to positive expression of ES cell markers, we also found 17 neural markers that were expressed by neural cells with little or no expression in ES cells and the liver (see list in Fig. 6C). Some of them were easily identified in row 5 (as indicated by the long arrow in Fig. 6A). RT-PCR analysis for expression of neural markers was in agreement with the array results (Fig. 6B). Interestingly glial markers present on

image profiles obtained by these two methods. The LPR method (71%, 184/260) also reveals higher detection rate than MMLV RT method (31%, 80/260). Short arrow: spot for integrin b5. Underline: four positive controls in triplicate. (B) Summarizes results comparing array data and RT-polymerase chain reaction (PCR) amplification. Both methods show high agreement (96-98%) between positive results in array and RT-PCR. The false-positive rate is 2.5-4.3%. Tables in (C) summarize the results of positive and negative hybridization from two experiments. A relative intensity of each spot was calculated and plotted for these repeated experiments. These scatter plots are shown in (D). The data suggest a larger variability for probe prepared by the LPR method compared with MMLV RT labeled probe. Further analysis of expression of five genes by semiquantitative RT-PCR is shown in (E). Results from this RT-PCR is in agreement with those obtained by MMLV RT method (see underline in A).

Fig. 5. Cross-hybridization test. cDNA fragments of 17 genes, as indicated in (A), were biotin-labeled, pooled, and used to probe the array. (A) Amplification quality of the PCR amplified and labeled probes. (B) An array image profile obtained by hybridization of the array with the mixture of 17 labeled probes. Location of each gene in array is indicated in bold letter in Fig. 1. Spots for FGF4 and NCAM2 were duplicated, and genes for Rpl13a, Actb, and GAPDH were spotted in triplicates. The results clearly indicate no cross hybridization using these 17 randomly genes. Spot intensities for genes present as duplicates and triplicates were also measured by using ImageQuan 5.2 and are presented in panel C. The data suggest a high reproducibility for duplicates and triplicates. CV: coefficient variance; SD/mean.

Fig. 5. Cross-hybridization test. cDNA fragments of 17 genes, as indicated in (A), were biotin-labeled, pooled, and used to probe the array. (A) Amplification quality of the PCR amplified and labeled probes. (B) An array image profile obtained by hybridization of the array with the mixture of 17 labeled probes. Location of each gene in array is indicated in bold letter in Fig. 1. Spots for FGF4 and NCAM2 were duplicated, and genes for Rpl13a, Actb, and GAPDH were spotted in triplicates. The results clearly indicate no cross hybridization using these 17 randomly genes. Spot intensities for genes present as duplicates and triplicates were also measured by using ImageQuan 5.2 and are presented in panel C. The data suggest a high reproducibility for duplicates and triplicates. CV: coefficient variance; SD/mean.

A Image Profiles Q RT-PCR

A Image Profiles Q RT-PCR

Q Comparison between Array and RT-PCR

Gene

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0.20

0.13

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080

037

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It«a6

0J9

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067

0,21

0.19

I"ou5Tl

0.»

0.21

014

023

0,15

0,21

Soxi

0.15

0.13

007

098

0.71

013

Ijfr

0 08

007

0.29

7/p42

0,40

006

003

Gem2

0.12

015

025

Prdr

0.16

0.23

009

Nafl-2

0.38

0.83

064

010

0.14

007

PraQauton

.\kiplb

0.»

0-77

030

009

0.19

004

TuhbJ

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0.13

0.17

0-55

0.85

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Cdial 111

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0.24

0.06

a2i

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0.14

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0.04

0.20

0.15

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0,43

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Cnvi2

0.00

0.13

0,0)

034

0,63

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CdM

001

001

0.08

023

0.49

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Niiml

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001

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Ntual

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0.42

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0.CM

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0.28

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Fig. 6. Evaluation of the array by using different types of tissues. Total RNAs (3 |g/membrane) derived from mouse D3 ES cells, rat E14.5 neural tubes and adult rat liver were used to make biotin dUTP-labeled cDNA probes with the moloney murine leukemia virus reverse transcriptase (RT) method. The array filters were hybridized with these probes and their images were recorded. (A) A representative image profile of three experiments. A short arrow indicates ES markers in the first row, whereas a longer arrow points to neural markers in the fifth row. (B) An image of RT- polymerase chain reaction (PCR) confirmation. Amounts of cDNAs from different tissues were adjusted to be nearly equal by using GAPDH as a control. (C) A summarized result derived from the array analysis and RT-PCR. Relative intensities of each gene expression relative to that of GAPDH were determined and are presented. 1, mouse D3 cells; 2, E14.5 rat NTs; 3, adult rat liver.

Table 2

A Partial List of Sources for Large Scale Genome Microarrays

Affymetrix; New York, NY Yale University; New Haven, CT Agilent; Foster City, CA MWG Biotech; High Point, NC

Amersham; Piscataway, NJ

NIAID Microarray Research Facility, NIH; Rockville, MD

NCI LMT microarray laboratory, NIH; Rockville, MD Qiagen; Valencia, CA Compugen; Jamesburg, NJ

http://www.affymetrix.com/products/arrays/index.affx

http://keck.med.yale.edu/dna_arrays.htm

www.chem.agilent.com/Scripts/pds.asp?lPage=2433

http://www.mwg-biotech.com/html/d_arrays/ d_catalog_arrays.shtml

http://www5.amershambiosciences.com/APTRIX/ upp01077.nsf/Content /codelink_human_bioarrays? OpenDocument&hometitle=codelink

http://www.niaid.nih.gov/dir/services/rtb/newmicro/ overview.asp

http://web.ncifcrf.gov/rtp/LMT/Microarray/default.asp

http://omad.qiagen.com/download/index.php

http://www.labonweb.com/chips/libraries.html

the array did not hybridize to probe prepared from neural cells at E14.5. It has been shown that glial progenitor cells appear late compared to neuronal progenitors in neural tubes (12). The stem array analysis also failed to detect glial markers, such as S100P, and GFAP, at this stage. Thus, this stem cell array can distinguish stem cells and progenitor cells at different development stages.

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