Grown for 13 days in growth chamber (16h/8h light/darkness, 50% humidity, 28°C, approx 40 µmol/s-1/m-2) in soil. BS cells collected from second leaves of 3rd leaf emerging stage seedlings.
Extracted molecule
total RNA
Extraction protocol
M protoplasts were isolated as previously described with slight modification; only second leaf blades were used. For full protocol see Markelz, N.H., Costich, D.E., and Brutnell, T.P. (2003). Photomorphogenic responses in maize seedling development. Plant Physiol 133, 1578-1591. RNA was isolated as in Sheehan, M.J., Farmer, P.R., and Brutnell, T.P. (2004). Structure and expression of maize phytochrome family homeologs. Genetics 167, 1395-1405.
Label
Cy5
Label protocol
Followed Genisphere Array 900MPX instructions using buffer 6. cDNA hybridization overnight at 55°C. Washed slides in 2X SSC, 0.5%SDS; 0.5X SSC; 0.05X SSC for 5 min each. First wash buffer was used at 55°C.
Grown for 13 days in growth chamber (16h/8h light/darkness, 50% humidity, 28°C, approx 40 µmol/s-1/m-2) in soil. BS cells collected from second leaves of 3rd leaf emerging stage seedlings.
Extracted molecule
total RNA
Extraction protocol
M protoplasts were isolated as previously described with slight modification; only second leaf blades were used. For full protocol see Markelz, N.H., Costich, D.E., and Brutnell, T.P. (2003). Photomorphogenic responses in maize seedling development. Plant Physiol 133, 1578-1591. RNA was isolated as in Sheehan, M.J., Farmer, P.R., and Brutnell, T.P. (2004). Structure and expression of maize phytochrome family homeologs. Genetics 167, 1395-1405.
Label
Cy3
Label protocol
Followed Genisphere Array 900MPX instructions using buffer 6. cDNA hybridization overnight at 55°C. Washed slides in 2X SSC, 0.5%SDS; 0.5X SSC; 0.05X SSC for 5 min each. First wash buffer was used at 55°C.
Hybridization protocol
Followed Genisphere Array 900MPX instructions using buffer 6. cDNA hybridization overnight at 55°C. Washed slides in 2X SSC, 0.5%SDS; 0.5X SSC; 0.05X SSC for 5 min each. First wash buffer was used at 55°C.
Scan protocol
See: Sawers, R.J., Liu, P., Anufrikova, K., Hwang, J.T., and Brutnell, T.P. (2007). A multi-treatment experimental system to examine photosynthetic differentiation in the maize leaf. BMC Genomics 8, 12.
Description
The mutant hcf136 lacks functional PSII components and granal stacking in its mesophyll (M) plastids. This severe disruption in photosynthetic functionality may result in changes to transcriptional profiles of spatially regulated C4 photosynthetic genes. As such, the M cell transcriptome of this mutant was assayed in comparison to its wild-type siblings using microarray analysis. M protoplasts were isolated by enzymatic digestion from the second leaves of wild-type and hcf136 plants at the third-leaf emerging stage of growth. Total RNA was isolated from the M cells, and M transcript profiles were compared between wild-type and hcf136 mutants using six biological replicates in a two-label direct comparison. The microarray experiments were performed using the Array 900MPX Expression Array Detection Kit (Genisphere), according to the prescribed company protocol.
Data processing
Lowess normalization. See: Sawers, R.J., Liu, P., Anufrikova, K., Hwang, J.T., and Brutnell, T.P. (2007). A multi-treatment experimental system to examine photosynthetic differentiation in the maize leaf. BMC Genomics 8, 12.
