Agilent eBook - 20

Best Practices with an Agilent Parallel CE System

10,000

8,000

RQN = 9.7
28S/18S = 1.9

18S

7,000
6,000
2,000
4,000

3,000

ProSize uses the RNA quality number (RQN) as a quality metric for total
RNA samples. ProSize considers the entire electropherogram including the
5S and fast region where the small RNA separates, as well as the small
and large ribosomal peaks, the baseline resolution between the peaks, the
ratio of the small and large ribosomal peaks, and the degradation in front
of the small ribosomal peak when calculating the RQN. The 5200 Fragment Analyzer system can easily distinguish between small RNA fragments
and degradation in the small RNA region, enabling ProSize to assign an
accurate RQN. The RQN is based on a scale from 1 to 10 where 1 represents completely degraded total RNA and 10 represents intact total RNA.
Universal mouse reference total RNA was separated on the 5200 Fragment
Analyzer system with the Agilent HS RNA kit (15 nt) (Figure 2). Freshly
prepared total RNA had a ribosomal ratio of 1.9 and an RQN of 9.7, while
the same sample subjected to 70 °C for 10 minutes reported a ribosomal

6,500

6,000

4,000

3,000

2,000

1,500

1,000

200

500

2,000

7,000

| GENengnews.com

28S

9,000

RQN

Size (bp)

B
18S

RQN = 5.9
28S/18S = 0.1

6,000
5,500
4,500

4,000
3,500
28S

3,000
2,500

6,000

4,000

3,000

2,000

1,500

1,000

500

2,000
15

RFU

5,000

200

As a dynamic quality metric, the GQN brings objective analysis to the
quality assessment of formalin-fixed, paraffin-embedded (FFPE) gDNA
samples. FFPE DNA undergoes substantial chemical modifications during
formalin fixation, including cross-linking and fragmentation. Utilizing the
5200 Fragment Analyzer system, Illumina developed three guidelines to
follow based on the GQN value when preparing an FFPE library: (1) minimal
DNA input into library preparation; (2) number of PCR cycles; and (3) the
amount of library used in enrichment2. Successful and reliable library preparations and sequencing results can be achieved with the GQN metrics.

12,000

RFU

Femto Pulse system with the Agilent Genomic DNA 165 kb kit. The size
threshold for these samples was set at 30,000 bp and reported a lower
GQN for the smaller sized samples compared to the larger sized gDNA, as
expected due to the varying size. The DQN and GQN values allow for fast
and easy assessment of the quality of any type of DNA sample.

Size (bp)

Figure 2. Universal mouse reference total RNA separated on the Agilent 5200 Fragment Analyzer system
with the Agilent HS RNA kit (15 nt). (A) 0 minutes at 70 °C, RQN = 9.7. (B) 10 minutes at 70 °C, RQN = 5.9.
LM = lower marker.

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Agilent eBook

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