RP23-321N22.11 UP in 'Nrl-/-' vs 'wild type'
Ube4bos2 DOWN in 'Nrl-/-' vs 'wild type'
Hist1h1c DOWN in 'Nrl-/-' vs 'wild type'
1700001L05Rik DOWN in 'Nrl-/-' vs 'wild type'
RP24-421N21.1 DOWN in 'Nrl-/-' vs 'wild type'
RP24-217M7.4 DOWN in 'Nrl-/-' vs 'wild type'
4932418E24Rik DOWN in 'Nrl-/-' vs 'wild type'
E030019B06Rik DOWN in 'Nrl-/-' vs 'wild type'
AA413626 DOWN in 'Nrl-/-' vs 'wild type'
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRTÃ¢ÂÂPCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods: Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl-/-) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: BurrowsâÂÂWheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRTÃ¢ÂÂPCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and NrlâÂÂ/âÂÂ mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRTÃ¢ÂÂPCR. RNA-seq data had a linear relationship with qRTÃ¢ÂÂPCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and NrlâÂÂ/âÂÂ retina, with a fold change âÂÂ¥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRTÃ¢ÂÂPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Retinal mRNA profiles of 21-day old wild type (WT) and Nrl-/- mice were generated by deep sequencing, in triplicate, using Illumina GAIIx.
2210406O10Rik UP in 'Nrl-/-' vs 'wild type'
2900052L18Rik UP in 'Nrl-/-' vs 'wild type'
I830134H01Rik DOWN in 'Nrl-/-' vs 'wild type'
A330094K24Rik DOWN in 'Nrl-/-' vs 'wild type'
Cyp4f41-ps DOWN in 'Nrl-/-' vs 'wild type'
1700030J22Rik DOWN in 'Nrl-/-' vs 'wild type'
8030423F21Rik UP in 'Nrl-/-' vs 'wild type'
1700010L04Rik DOWN in 'Nrl-/-' vs 'wild type'
1110035H17Rik UP in 'Nrl-/-' vs 'wild type'
RP23-80O10.1 DOWN in 'Nrl-/-' vs 'wild type'
A930031H19Rik DOWN in 'Nrl-/-' vs 'wild type'
Arhgap10 DOWN in 'Nrl-/-' vs 'wild type'
Epb4.1l2 DOWN in 'Nrl-/-' vs 'wild type'
D1Ertd622e DOWN in 'Nrl-/-' vs 'wild type'
(Factor value) GENOTYPE/wild type genotype
Hist1h2be DOWN in 'Nrl-/-' vs 'wild type'
4930578M01Rik UP in 'Nrl-/-' vs 'wild type'
RP24-166N8.2 DOWN in 'Nrl-/-' vs 'wild type'
3425401B19Rik DOWN in 'Nrl-/-' vs 'wild type'
4833417C18Rik UP in 'Nrl-/-' vs 'wild type'
Tmem255b DOWN in 'Nrl-/-' vs 'wild type'
1700067P10Rik UP in 'Nrl-/-' vs 'wild type'
2310030G06Rik UP in 'Nrl-/-' vs 'wild type'
4930465K10Rik DOWN in 'Nrl-/-' vs 'wild type'
4921507G05Rik DOWN in 'Nrl-/-' vs 'wild type'
Ppargc1b DOWN in 'Nrl-/-' vs 'wild type'
A630095N17Rik DOWN in 'Nrl-/-' vs 'wild type'
6430562O15Rik DOWN in 'Nrl-/-' vs 'wild type'
4933406J10Rik DOWN in 'Nrl-/-' vs 'wild type'
RP23-45G16.6 DOWN in 'Nrl-/-' vs 'wild type'
A630076J17Rik DOWN in 'Nrl-/-' vs 'wild type'
B230319C09Rik DOWN in 'Nrl-/-' vs 'wild type'
5330416C01Rik UP in 'Nrl-/-' vs 'wild type'
4930458D05Rik UP in 'Nrl-/-' vs 'wild type'
2010107G12Rik UP in 'Nrl-/-' vs 'wild type'
5430419D17Rik UP in 'Nrl-/-' vs 'wild type'
4933439K11Rik UP in 'Nrl-/-' vs 'wild type'
4933433G15Rik DOWN in 'Nrl-/-' vs 'wild type'
2500002B13Rik DOWN in 'Nrl-/-' vs 'wild type'
5730522E02Rik UP in 'Nrl-/-' vs 'wild type'
Hist1h1e DOWN in 'Nrl-/-' vs 'wild type'
RP23-440M14.1 UP in 'Nrl-/-' vs 'wild type'
A930038B10Rik UP in 'Nrl-/-' vs 'wild type'
RP23-285F13.1 UP in 'Nrl-/-' vs 'wild type'
3222401L13Rik DOWN in 'Nrl-/-' vs 'wild type'
1700001K19Rik DOWN in 'Nrl-/-' vs 'wild type'
2010001A14Rik DOWN in 'Nrl-/-' vs 'wild type'
Slc25a25 DOWN in 'Nrl-/-' vs 'wild type'
0610038B21Rik DOWN in 'Nrl-/-' vs 'wild type'
RP23-282G20.2 DOWN in 'Nrl-/-' vs 'wild type'
E130018N17Rik DOWN in 'Nrl-/-' vs 'wild type'
RP24-240G4.2 UP in 'Nrl-/-' vs 'wild type'
4930581F22Rik DOWN in 'Nrl-/-' vs 'wild type'
1520401A03Rik DOWN in 'Nrl-/-' vs 'wild type'
C230014O12Rik UP in 'Nrl-/-' vs 'wild type'
A930006I01Rik DOWN in 'Nrl-/-' vs 'wild type'
Pitpnm2os2 DOWN in 'Nrl-/-' vs 'wild type'
1810010K12Rik DOWN in 'Nrl-/-' vs 'wild type'
1500002C15Rik DOWN in 'Nrl-/-' vs 'wild type'
Ube4bos1 DOWN in 'Nrl-/-' vs 'wild type'
Selenbp1 DOWN in 'Nrl-/-' vs 'wild type'
Slc22a30 DOWN in 'Nrl-/-' vs 'wild type'
RP23-158L19.7 DOWN in 'Nrl-/-' vs 'wild type'
3010001F23Rik DOWN in 'Nrl-/-' vs 'wild type'
D430019H16Rik UP in 'Nrl-/-' vs 'wild type'
1700101E01Rik DOWN in 'Nrl-/-' vs 'wild type'
E230008N13Rik DOWN in 'Nrl-/-' vs 'wild type'
3110070M22Rik DOWN in 'Nrl-/-' vs 'wild type'
Arhgap19 DOWN in 'Nrl-/-' vs 'wild type'
AA986860 DOWN in 'Nrl-/-' vs 'wild type'
4932442L08Rik DOWN in 'Nrl-/-' vs 'wild type'
Arhgap27os3 UP in 'Nrl-/-' vs 'wild type'
Rplp2-ps1 DOWN in 'Nrl-/-' vs 'wild type'
2310014L17Rik UP in 'Nrl-/-' vs 'wild type'
4930445N18Rik DOWN in 'Nrl-/-' vs 'wild type'
Slc22a28 DOWN in 'Nrl-/-' vs 'wild type'
1810041L15Rik UP in 'Nrl-/-' vs 'wild type'
RP24-89G21.1 UP in 'Nrl-/-' vs 'wild type'
AC118721.1 UP in 'Nrl-/-' vs 'wild type'
4933415A04Rik UP in 'Nrl-/-' vs 'wild type'
A930009A15Rik UP in 'Nrl-/-' vs 'wild type'
1110007C09Rik DOWN in 'Nrl-/-' vs 'wild type'
RP23-408A1.2 DOWN in 'Nrl-/-' vs 'wild type'
5033430I15Rik DOWN in 'Nrl-/-' vs 'wild type'
1700003M02Rik DOWN in 'Nrl-/-' vs 'wild type'
1700071M16Rik DOWN in 'Nrl-/-' vs 'wild type'
D430040D24Rik DOWN in 'Nrl-/-' vs 'wild type'
RP23-245G9.1 DOWN in 'Nrl-/-' vs 'wild type'
D330050G23Rik UP in 'Nrl-/-' vs 'wild type'
Als2cr11 DOWN in 'Nrl-/-' vs 'wild type'
Tnfsf13b DOWN in 'Nrl-/-' vs 'wild type'
D930007P13Rik UP in 'Nrl-/-' vs 'wild type'
4930447C04Rik UP in 'Nrl-/-' vs 'wild type'
C230088H06Rik UP in 'Nrl-/-' vs 'wild type'
A930018P22Rik DOWN in 'Nrl-/-' vs 'wild type'
A530058N18Rik DOWN in 'Nrl-/-' vs 'wild type'
RP24-89C18.2 DOWN in 'Nrl-/-' vs 'wild type'
RP23-185G9.3 DOWN in 'Nrl-/-' vs 'wild type'
Tmem181b-ps UP in 'Nrl-/-' vs 'wild type'
4930526A20Rik DOWN in 'Nrl-/-' vs 'wild type'
D630003M21Rik DOWN in 'Nrl-/-' vs 'wild type'
Fam160a1 DOWN in 'Nrl-/-' vs 'wild type'
1700125G02Rik UP in 'Nrl-/-' vs 'wild type'
RP23-153H17.1 DOWN in 'Nrl-/-' vs 'wild type'
RP23-435I22.4 DOWN in 'Nrl-/-' vs 'wild type'
4931415C17Rik DOWN in 'Nrl-/-' vs 'wild type'
C430042M11Rik DOWN in 'Nrl-/-' vs 'wild type'
BC025920 DOWN in 'Nrl-/-' vs 'wild type'
4933427G17Rik DOWN in 'Nrl-/-' vs 'wild type'
BC065403 DOWN in 'Nrl-/-' vs 'wild type'
Arhgap27os1 UP in 'Nrl-/-' vs 'wild type'
2610034M16Rik DOWN in 'Nrl-/-' vs 'wild type'
RP24-79I19.2 UP in 'Nrl-/-' vs 'wild type'
A930024N18Rik UP in 'Nrl-/-' vs 'wild type'
Marveld3 DOWN in 'Nrl-/-' vs 'wild type'
RP23-473M13.3 UP in 'Nrl-/-' vs 'wild type'
4930554H23Rik UP in 'Nrl-/-' vs 'wild type'
0610005C13Rik UP in 'Nrl-/-' vs 'wild type'
1810009A15Rik DOWN in 'Nrl-/-' vs 'wild type'
4930470G03Rik UP in 'Nrl-/-' vs 'wild type'
F420014N23Rik DOWN in 'Nrl-/-' vs 'wild type'
Rpl31-ps14 UP in 'Nrl-/-' vs 'wild type'
2610035F20Rik UP in 'Nrl-/-' vs 'wild type'
RP23-431O5.2 UP in 'Nrl-/-' vs 'wild type'
4930555M17Rik DOWN in 'Nrl-/-' vs 'wild type'
A930007I19Rik DOWN in 'Nrl-/-' vs 'wild type'
A930011G23Rik DOWN in 'Nrl-/-' vs 'wild type'
4933416I08Rik UP in 'Nrl-/-' vs 'wild type'
RP23-53N22.1 DOWN in 'Nrl-/-' vs 'wild type'
A930004D18Rik DOWN in 'Nrl-/-' vs 'wild type'
Slc25a29 DOWN in 'Nrl-/-' vs 'wild type'
Traf3ip3 DOWN in 'Nrl-/-' vs 'wild type'
BB218582 DOWN in 'Nrl-/-' vs 'wild type'
4930583K01Rik DOWN in 'Nrl-/-' vs 'wild type'
Smarca5-ps DOWN in 'Nrl-/-' vs 'wild type'
C130080G10Rik DOWN in 'Nrl-/-' vs 'wild type'
1700023L04Rik UP in 'Nrl-/-' vs 'wild type'
C030048H21Rik DOWN in 'Nrl-/-' vs 'wild type'
Rps2-ps10 DOWN in 'Nrl-/-' vs 'wild type'
RP23-52N17.1 DOWN in 'Nrl-/-' vs 'wild type'
4930533B01Rik UP in 'Nrl-/-' vs 'wild type'
A230060F14Rik UP in 'Nrl-/-' vs 'wild type'
A930012L18Rik UP in 'Nrl-/-' vs 'wild type'
BC037032 DOWN in 'Nrl-/-' vs 'wild type'
E230016M11Rik DOWN in 'Nrl-/-' vs 'wild type'
St6galnac5 DOWN in 'Nrl-/-' vs 'wild type'
A930018M24Rik UP in 'Nrl-/-' vs 'wild type'
BC030870 DOWN in 'Nrl-/-' vs 'wild type'
Slc22a29 DOWN in 'Nrl-/-' vs 'wild type'
RP23-101D11.2 DOWN in 'Nrl-/-' vs 'wild type'
RP23-8J15.5 UP in 'Nrl-/-' vs 'wild type'
A930041C12Rik DOWN in 'Nrl-/-' vs 'wild type'
C4bp-ps1 DOWN in 'Nrl-/-' vs 'wild type'
4930551O13Rik DOWN in 'Nrl-/-' vs 'wild type'
1700007K13Rik DOWN in 'Nrl-/-' vs 'wild type'