TB-Synthesis and Labeling of cDNA SKlab 01; 1. Bring 0.5-5 ?g RNA (Typically 3 ?g) to 11 ?l and add 2 ?l (~2 mg/ml) random hexamers. 2. Heat 10 min at 65C (or 2 min at 98C), snap cool on ice. 10 rxns 20 rxns 3. Add 11?l 5.0 ?l 5X First-Strand buffer (51) (102) 2.5 ?l 100 mM DTT (25.5) (51) 2.0 ?l dNTP (5 mM A,G,C and 0.2 mM T) (20.5) (41) 1.5 ?l Cy3 or Cy5 (Typically use Cy5 for sample that should change) (15.3) (30.6) Then add 1.2-1.8 ?l Stratascript or 0.8-1.2 ?l Superscript II RTase. 4. Incubate 10m at 25C followed by 90m at 42C in PCR machine. Can freeze or leave at 4C o/n.; Protocol Type = Labeling; Performer: Martin,,Voskuil
Growth Conditions; Parameters that describe the conditions under which the biological sample was grown or propagated; Protocol Type = Growth Conditions; Parameter Starting OD = 0.15; Parameter Elapsed Time = 40 min; Parameter Growth Temperature = 37oC; Parameter Culture Synchrony = Unsynchronized; Parameter Growth Vessel Volume = 250 ml; Parameter Media = Middlebrook 7H9 supplemented with albumin-dextrose complex (ADC) and 0.01% Tween 80; Performer: Martin,,Voskuil
Extracted molecule
total RNA
Extraction protocol
not provided TB-RNA Isolation and Purification SKlab 01; 1. Centrifuge 20-30 ml of early- to mid-log culture (O.D. 0.1-0.2) 4 min at 3700 rpm, at RT-37C. Pipette off supernatant and immediately freeze on dry ice and store at ?80C. 2. Add 1 ml Trizol (Gibco-BRL) to each of 4-8 pellets, suspend first pellet by vortexing,while other pellets are still frozen. Add suspension to 0.4 ml glass beads in a 2 ml screw cap tube. 3. Shake 30s at maximum speed in bead beater. Suspend next pellet and add to glass beads. Apply next sample to bead beater. Repeat bead beating two more times with each sample.Continue to periodically invert samples for at least 5 min in Trizol. 4. Centrifuge samples 45s at max speed, remove Trizol solution to a 2 ml screw cap tube containing half of the Heavy Phase Lock Gel I, supplied by 5 Prime 3 Prime, Inc. in 1.5 ml tubes and transferred with sterile stick to 2 ml tubes, and 300 ?l Chloroform:isoamyl alcohol (24:1). Invert rapidly for 15s, and continue inverting periodically for 2 min.a 5. Centrifuge 5-10m, remove aqueous layer (540 ?l) and add to a 1.5 ml tube containing 270 ?l isopropanol then add 270 ?l high salt solution (0.8M Na Citrate, 1.2M NaCl). Invert several times and spray with Staphene and remove from the P3. Typically ppted at 4C O/N. 6. Centrifuge 10m at 4C and remove isopropanol. Add 1 ml 75% EtOHb, invert several times and centrifuge 5m. 7. Remove EtOH by aspiration. Then, dry under vacuum for 2 min [Do not over dry]. 8. Suspend RNA in 90 ml RNase free water (don?t suspend in DNaseI buffer), may need to heat 10m at 55-60C to dissolve RNA. (Optional: obtain RNA concentration) 9. Add 10 ?l 10X DNaseI buffer to RNA (use no more than 80 ?g RNA) and add 4 ?l DNaseI (Ambion). Incubate 30m at 37C. Qiagen RNeasy purification. 1. Add 350 ?l RLT buffer (add 10 ?l BME to 1 ml RLT before using) and vortex. Add 260 ?l 95% EtOH (or 250 ?l 100% EtOH) to each sample and vortex. 2. Add to RNeasy spin column, centrifuge 15s, transfer column to a new 2 ml collection tube. Add 500 ?l RPE, centrifuge 15s, discard flow-through, add 500 ?l additional RPE and centrifuge 2m. If column still wet on sides, remove wash solution from tube and spin 1m to dry. 3. Transfer to a 1.5 ml collection tube, elute with 40 ?l RNase free water, centrifuge 1m. 4. Determine RNA concentration with A260/A280 readings. Dilute 1 ?l in 199 ?l TE. [200(dilution factor)x 40 ?g/A260x A260 = ?g/ml]. 5. Run 1 ?l RNA on 2% Agarose TAE gel. Run gel 45m at 100 volts. aIf Phase Lock Gel is not used, decrease Chloroform:isoamyl alcohol to 200?l. The yield will be less. Be careful not to remove any of the interface layer. bUsing 100% EtOH should be avoided in all steps of preparation of RNA intended for array analysis. Instead use 95% EtOH in preparation of solutions. Benzene contamination may fluoresce. cInstead of using the RNeasy purification, the DNaseI can be inactivated at 65-70C for 15m and then EtOH precipitated. The RNeasy purification may not be necessary in all applications. We find the purification gives more consistent results and is less time consuming, compared to precipitation. In addition, other purifications can be used. Note: The RNeasy column will remove much of the small tRNA and degraded RNA.; Protocol Type = Extract preparation; Performer: Martin,,Voskuil
Label
Cy5
Label protocol
TB-Slide Preparation SKlab 01; 1. Prepare NaOH-ethanol solution ? dissolve NaOH in ddH2O 70 g / 280 mL 175 g / 700mL 200 g / 800 mL ? stir until completely dissolved ? add 95% ethanol 420 mL 1050 mL 1200mL ? stir until completely mixed ? if solution remains cloudy, add water until clear 2. Place slides in metal slide racks (30 slides/rack). Do not use defective slides. 3. Soak slides in the NaOH:EtOH:ddH2O solution for 2 hours with gentle rotation. 4. Rinse extensively with dH2O: ? rinse each unit (slide/rack/container) vigorously with dH2O for 5 min ? place slide racks in a large clean glass container, and tilt the container slightly for constant water flow. ? wash under running water for 30 minutes. ? do not allow the slides to dry at any time. It is critical to remove all traces of NaOH:EtOH. 5. Prepare poly-L-lysine solution in plastic container. ? 100 mL tissue culture PBS 800 mL Milli-Q water 100 mL poly-L-Lysine ? We bring up the volume to about 1050 mL with Milli-Q water in order to submerge 3 racks of slides. ? Mix well and split into 3 plastic containers. 6. Soak the slides in lysine solution for 45 min with shaking. Be sure to use a plastic container, because poly-L-lysine adheres to glass. Poly-L-Lysine solution may be reused. Keep the other slide filled racks in dH2O, while the first 3 are being coated. 7. After the lysine coating, rinse the slides by gently plunging up and down in 2 different changes of water. Spin dry 5' at 600 rpm. Place paper towels below rack to absorb liquid. 8. Store slides in a dessicator for 3 weeks prior to use. Slides that are older than 3 months may result in faint printing and higher background. Be sure to clean racks and containers thoroughly after each use. Built-up poly-L-Lysine on the sides of the containers may cause problems.; Protocol Type = Treatment; Performer: Martin,,Voskuil Labeling - Channel 2; Methods for labeling extracted molecules that are used in hybridizations and scanned in Channel 2 (red); Protocol Type = Labeling; Parameter Amount of extract labeled = 1.5ug of total RNA; Parameter Type of label = Cy5; Performer: Martin,,Voskuil
Median intensities of background pixels of Cy3.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2B_MEDIAN
Median intensities of background pixels of Cy3.; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1D_MEAN
The mean feature pixel intensity with the median background subtracted (channel 1).; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2D_MEAN
The mean feature pixel intensity with the median background subtracted (channel 2).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1B_MEAN
Mean intensities of background pixels of Cy3.; Type: integer; Scale: linear_scale; Background
CH2B_MEAN
Mean intensities of background pixels of Cy5.; Type: integer; Scale: linear_scale; Background
PERGTBCH1I_1SD
The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale
PERGTBCH2I_1SD
The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 635 nm.; Type: integer; Scale: linear_scale
PIX_RAT2_MEDIAN
Contains median of Ch2PI-CH2B/Ch1PI-CH1B where Ch1PI & Ch2PI represent single pixel intensities.; Type: float; Scale: linear_scale
TOT_SPIX
Count of the number of pixels in the spot.; Type: integer; Scale: linear_scale
TOT_BPIX
Number of background pixels.; Type: integer; Scale: linear_scale
REGR
The regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.; Type: float; Scale: linear_scale
CORR
The correlation between channel1 (Cy3) & Channel 2 (Cy5) pixels within the spot, and is a useful quality control parameter. Generally, high values imply better fit & good spot quality.; Type: float; Scale: linear_scale
User defined spot flag (default 0).; Type: integer; Scale: linear_scale
CH2IN_MEAN
Normalized value of mean Channel 2 (usually 635 nm) intensity (CH2I_MEAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH2BN_MEDIAN
Normalized value of median Channel 2 (usually 635 nm) background (CH2B_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background
CH2DN_MEAN
Normalized value of mean Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
RAT2N_MEAN
Type: float; Scale: linear_scale
RAT1N_MEAN
Ratio of the means of Channel 1 (usually 532 nm) intensity to normalized Channel 2 (usually 635 nm) intensity with median background subtracted (CH1D_MEAN/CH2DN_MEAN). Channel 1/Channel 2 ratio normalized or Green/Red ratio normalized.; Type: float; Scale: linear_scale
VALUE
Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEAN)].; Type: float; Scale: log_base_2
