Feasibility of RNA and DNA Extraction from Fresh Pipelle and Archival Endometrial Tissues for Use in Gene Expression and SNP Arrays
Table 2
RNA quality metrics and assay performance.
Benign
Complex hyperplasia
Hyperplasia with atypia
Cancer
FF Pipelle
FF Curettage
FFPE
Normal
Involved
Normal
Involved
Normal
Involved
RNA Extraction
Number successfully extracted (over number attempted)*
7/7
6/8
8/8
5/6
6/6
6/6
6/6
7/7
12/12
Mean RNA yield† (g) (range)
35.4 (20.9–62.0)
32.7 (25.1–50.4)
11.6 (2.7–26)
16.4 (5.7–24.9)
17.1 (1.6–48.5)
14.5 (6.9–21.4)
11.1 (8.0–17.8)
13.9 (6.1–43.1)
25.5 (11.1–39.7)
Mean 260/280 ratio† (range)
2.05 (2.01–2.08)
2.06 (2.05–2.09)
2.00 (1.9–2.0)
2.02 (1.93–2.05)
2.05 (1.91–2.1)
2.02 (1.97–2.05)
1.98 (1.8–2.03)
2.04 (1.93–2.09)
2.00 (1.9–2.1)
DASL Pre-qualification‡
Mean Ct threshold (range)
15.7 (15.0–16.4)
15.7 (15–18.7)
21.0 (18.0–25.4)
20.9 (19.6–22.5)
19.5 (18.2–20.7)
20.5 (19.3–21.3)
19.9 (20.0–21.7)
21.5 (19–24.7)
21.0 (18.5–27.6)
DASL Expression Array
Array pass rat
7/7
6/6
8/8
5/5
5/5
6/6
6/6
7/7
12/12
Average number of probes detected (of 24,526, )
18,640 (76.0%)
18,165 (74.1%)
17,197 (70.1%)
18,455 (75.2%)
18,244 (74.4%)
18,239 (74.4%)
18,446 (75.2%)
18,177 (74.1%)
17,648 (72%)
Average number of probes detected (of 24,526, )
17,064 (69.6%)
16,637 (67.8%)
15,640 (63.8%)
16,912 (69.0%)
16,846 (68.7%)
16,647 (67.9%)
16,823 (68.6%)
16,442 (67.0%)
15,874 (64.7%)
Average number of genes detected (of 18,401,
12,917 (70.2%)
12,630 (68.6%)
11,931 (64.8%)
12,434 (67.6%)
12,580 (68.4%)
12,642 (68.7%)
12,645 (68.7%)
12,348 (67.1%)
11,930 (64.8%)
Average Signal
7,436
8,495
6,630
6,906
6,944
7,202
6,921
6,703
6,725
191
188
184
179
184
176
179
178
192
Replicate Concordance
Number of replicates run on array
3
4
4
3
3
3
3
5
5
Correlation of expression levels
0.99
0.99
0.99
0.99
0.99
0.98
0.99
0.99
0.98
Probe concordance,
0.97
0.98
0.96
0.96
0.96
0.95
0.96
0.96
0.94
Probe concordance,
0.98
0.98
0.97
0.96
0.97
0.96
0.96
0.96
0.94
Extractions were defined as successful if the yield was >1.2 g and the 260/280 ratio was between 1.80 and 2.20, as pure RNA has a 260/280 ratio of 2.0. Excluding the three samples that had yields <1.2 g. A Ct threshold of 29 or below qualifies RNA for the DASL array. All replicates also passed. In our high-throughput facility, the average number of genes detected is 10,000, the average p95 signal is 5,000, and the average signal-to-noise ratio is 40.
