Report Content

The following are examples of Eukaryotic 3'- expression report files for each QC step and a summary of the experiment result.

1st QC | 2nd and 3rd QC | 4th QC | summary | Example

  • 1st QC of RNA quality (include results of OD and Bioanalyzer)

Assigned no Number we assigned

Threshold
User User name

N/A
User Sample ID

Sample name that user give

 N/A
Sample type RNA type N/A
Submitted Date

Date of sample submission

 N/A

UV spectrophotometer (0.5 μl RNA + 3 μl 10mM Tris pH 8.0 buffer, by NanoDrop)
230 Value of OD230

0.02 - 75
260 Value of OD260 0.02 - 75
280 Value of OD280 0.02 - 75
340 Value of OD340, background 0.02 - 75
260/230 ratio OD260-340 / OD230-340 > = 1.8
260/ 280 ratio OD260-340 / OD280-340   > = 1.9
Original concentration (μg/μl)

Original concentration of total RNA or mRNA

 1.5 - 3
Agilent Bioanalyzer  (following three data come from Bioanalyzer）
28S peak > 18S peak It dependents on different species.

28S peak > 18S peak
28S / 18S ratio The ratio of 28S/18S > = 1.4
total area of (28S% + 18S %)

Total area of 28S% + 18S%

 > = 40

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  • Picture of Bioanalyzer analysis

  • 2nd and 3rd: QC for the efficiency of IVT and Fragmentation

IVT (In vitro transcription)
Date

Date of IVT reaction

N/A
IVT kit 3’- Amplification for IVT Labeling N/A
230 OD230 of cRNA 0.02 - 75
260 OD260 of cRNA 0.02 - 75
280 OD280 of cRNA 0.02 - 75
340 OD340 of cRNA, background 0.02 - 75
260/230 ratio OD260-340 / OD230-340 NA
260/280 ratio OD260-340 / OD280-340 >1.9
Original concentration (μg/μl)

Original concentration of cRNA

 > = 1.5
Major peak (nt)

The major peak of cRNA should be greater than 1000 nt at least in general.

 > = 1000
Fragmentation
Smear range (nt)

The fragmentized cRNA should be smear from 35 - 200 nt.

Btwn 35-200

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  • Picture of cRNA and fragment cRNA electrophoresis.

  • Hybridization, wash/stain and image processing record

Chip Hybridization
Date

Date of hybridization

 N/A
Chip type GeneChip type of usage N/A
Lot#

GeneChip lot no. GeneChips from the same lot are used for one user if available.

 N/A
Hybridization hours 16.5 hr N/A
Wash / Stain
Date

Date of washing / staining

 N/A
Protocol

Wash/stain protocol of Fluidic Station 450

 N/A
Image Data   (data analysis and produce after chip scanning)
Date

Date of image scanning

N/A
Grid aligned Aligned to four corners of chip OK
Borders stained Staining of the border of GeneChip for alignment OK

  • 4th QC of hybridization control

Report file

Sample assigned no.

N/A
Scaling factor

For eliminating the non-biological effect between GeneChips, it is necessary for signal normalization before data comparison. Scaling factor standardizes the average signal of each experiment to a TGT value. If the scaling factor with the same TGT value of compared GeneChip differs by more than 3-fold, it should be treated with caution.

N/A
TGT value

Default value was 500 (Rice and Soybean were 250), please see scaling factor.

 Def = 500
Background Value of chip background <100
Percentage of Present The percentage of significantly expressed probe sets > 30
AFFX-Actin 3’/ 5’ ratio The 3’/5’ ratio of housekeeping gene reveals the IVT efficiency. < 3, approx 1
AFFX-GAPDH 3’/ 5’ ratio The 3’/5’ ratio of housekeeping gene reveals the IVT efficiency.. < 3, approx 1
AFFX-r2-BIOB Spike-in controls which monitor the process of wash, stain and scanning. P or A
AFFX-r2-BIOC Spike-in controls which monitor the process of wash, stain and scanning. P
AFFX-r2-BIOD Spike-in controls which monitor the process of wash, stain and scanning. P
AFFX-r2-CRE Spike-in controls which monitor the process of wash, stain and scanning. P

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  • Picture of GeneChip scanning.

  • Summary of the QC report.

  1. QC result. (Summary all of the QC results.)

  2. TGT value and the scaling factor. (The TGT value and scaling factor will be reported for future reference.)

  3. Number for Present.  (It depends on species and treatment, etc.)

  4. Housekeeping Control. (The 3'/5' ratio of actin and GAPDH should not be over 3.)

  5. Spike Controls. (BioC, D, cre should be Present, but BioB may be Present or Absent.)

Updated at 2009/5/8