Objectives Objectives Objectives Objectives

5 downloads 0 Views 3MB Size Report
Lobaria pulmonaria contains a fungus, alga, and cyanobacteria. Objectives. 1) To optimise rehydration conditions for best RNA iso fungus. Lobaria pulmonaria ...
Photo: T. Booth, 2008.

Unlocking the secrets of optimal RNA isolation from Lobaria pulmonaria (L.) Hoffm. Jennifer Lawyer ([email protected]) and Michele Piercey-Normore Piercey Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, R3T 2N2

Introduction

Methods

1) To optimise rehydration conditions for best RNA isolation in Lobaria pulmonaria 2) To compare RNA quantity and quality before and after subjection to DNA removal 3) To determine the saturation point of PCR using small mitrochondrial subunit genes after cDNA synthesis

Results a

200.0 Mean Absolute RNA (ng/µL/g)

• A lichen is the result of a symbiotic relationship between an alga and a fungus. Lobaria pulmonaria contains a fungus, alga, and cyanobacteria in its symbiotic relationship. • Lichens are able to rehydrate and dehydrate due to high amounts of polyols (Nash 1996). L. pulmonaria cannot rehydrate and dehydrate as often as other lichens. • During rehydration, cell membranes are damaged, resulting in the leakage of polyols (Farrar 1976). This causes endonucleases (RNases) to be released, which break down the RNA in the cells. •RNAse activity increases during rehydration (de los Rios et al. 1996), making it hard to isolate RNA for research. • Low quality RNA may generate poor results (Imbeaud et al 2005), but little is known about the conditions required to yield good quality RNA. • Lichens cannot recover if the polyol concentration decreases below a critical point, which is species specific (Farrar 1976). • Lobaria pulmonaria is a charismatic foliose lichen commonly found in old growth forests (Brodo et al 2001), and is used as a bioindicator. This species has been chosen as a model organism by the international lichenological community and has been extensively studied from many perspectives: population genetics and dispersal, physiological interactions, conservation genetics, sexual reproductive capacity, and the genomes and transcriptomes of all three symbionts are currently being sequenced (DOE Joint Genome Project). •Methods to extract optimal DNA and RNA from this species are needed to enable extensive and efficient study of this complex species.

180.0 160.0 ab

120.0

ab

ab

ab

100.0 80.0

ab

ab

140.0

ab

ab

b

ab

b

b

60.0 40.0

. 0

A

1

2

3

4

5

6 7 8 Treatment

9

10

11

12

13

1.7 1.6 1.5 1.4

c

c

1.6

Treatment 8 is significantly higher in quality than treatments 3 and 6 for all samples.

1.4

RNA quantity cannot be used to predict quality.

0

1

2

4

3

5

6 7 8 Treatment

9

10 11 12 13

1.2

1.7

This relationship was seen both before and after DNA removal.

1.5

1.3 50

100

150

200

250

300

B

2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0

RNA quantity (ng/µL/g) bef ore DNA removal

RNA quantity (ng/µL/g)

y = 0.5748x + 0.6725 R² = 0.357 p value: